Transcription factors for increasing yield

ABSTRACT

The invention is directed to transgenic plants transformed with nucleic acids that encode a plant transcription factor that increases the transgenic plant&#39;s size and yield and/or delays flowering in the plant, and methods of using and producing the transgenic plants.

RELATED APPLICATION INFORMATION

The present patent application is a continuation-in-part application of prior U.S. patent application Ser. No. 09/713,994, filed Nov. 16, 2000, which is now abandoned, and the present patent application claims the benefit of the following three U.S. provisional patent applications: application Ser. No. 60/166,228 filed Nov. 17, 1999; application Ser. No. 60/197,899, filed Apr. 17, 2000; and application Ser. No. 60/227,439, filed Aug. 22, 2000. The entire contents of each of these applications are hereby incorporated by reference.

JOINT RESEARCH AGREEMENT

The claimed invention, in the field of functional genomics and the characterization of plant genes for the improvement of plants, was made by or on behalf of Mendel Biotechnology, Inc. and Monsanto Company as a result of activities undertaken within the scope of a joint research agreement, said agreement having been in effect on or before the date the claimed invention was made.

FIELD OF THE INVENTION

This invention relates to the field of plant biology. More particularly, the present invention pertains to compositions and methods for phenotypically modifying a plant.

BACKGROUND OF THE INVENTION

Transcription factors can modulate gene expression, either increasing or decreasing (inducing or repressing) the rate of transcription. This modulation results in differential levels of gene expression at various developmental stages, in different tissues and cell types, and in response to different exogenous (e.g., environmental) and endogenous stimuli throughout the life cycle of the organism.

Because transcription factors are key controlling elements of biological pathways, altering the expression levels of one or more transcription factors can change entire biological pathways in an organism. For example, manipulation of the levels of selected transcription factors may result in increased expression of economically useful proteins or metabolic chemicals in plants or to improve other agriculturally relevant characteristics such as to increase yield. Therefore, manipulating transcription factor levels in a plant offers tremendous potential in agricultural biotechnology for modifying a plant's traits such as improved yield from commercially important plant species.

One factor affecting yield is the number of plants that can be grown per acre. For crop species, planting or population density varies from a crop to a crop, from one growing region to another, and from year to year. Using corn as an example, the average prevailing density in 2000 was in the range of 20,000-25,000 plants per acre in Missouri, USA. A desirable higher population density (a measure of yield) would be at least 22,000 plants per acre, and a more desirable higher population density would be at least 28,000 plants per acre, more preferably at least 34,000 plants per acre, and most preferably at least 40,000 plants per acre. The average prevailing densities per acre of a few other examples of crop plants in the USA in the year 2000 were: wheat 1,000,000-1,500,000; rice 650,000-900,000; soybean 150,000-200,000, canola 260,000-350,000, sunflower 17,000-23,000 and cotton 28,000-55,000 plants per acre (Cheikh et al. (2003) U.S. Patent Application No. 20030101479). A desirable higher population density for each of these examples, as well as other valuable species of plants, would be at least 10% higher than the average prevailing density or yield.

The present invention provides novel transcription factors useful for modifying a plant's phenotype in desirable ways.

SUMMARY OF THE INVENTION

The present invention pertains to a transgenic plant having that has an improved trait relative to a control plant. The improved trait may include, for example, larger size, larger seeds, greater yield, darker green, increased rate of photosynthesis, more tolerance to osmotic stress, more drought tolerance, more heat tolerance, more salt tolerance, more cold tolerance, more tolerance to low nitrogen, early flowering, delayed flowering, more resistance to disease, more seed protein, and more seed oil relative to the control plant. The transgenic plant will generally comprise an expression vector that comprises a recombinant polynucleotide of the invention, that is, a nucleic acid sequence that encodes a polypeptide sequence that is related to any of SEQ ID NO: 110, 112, 116, 120, 124, 128, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 280, 284, 288, 292, 296, 299, 303, 306, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 401, 404, 406, 409, 413, 416, 419, 422, 425, 428, 431, 435, 439, 443, 447, 451, 454, 458, 462, 465, 468, 471, 475, 478, 482, 485, 489, 493, 497, 501, 505, 509, 512, 515, 519, 522, 526, 530, 534, 538, 542, 546, 550, 553, 557, 561, 565, 568, 571, 574, 577, 581, 585, 588, 591, 594, 597, 601, 605, 609, 613, 616, 620, 624, 628, 632, 636, 640, 644, 648, 652, 656, 660, 664, 667, 671, 674, 678, 682, 686, 689, 692, 696, 700, 704, 708, 712, 715, 719, 723, 727, 731, 734, 738, 741, 745, 749, 752, 756, 760, 762, 766, 770, 774, 778, 782, 786, 789, 793, 797, 801, 805, 809, 813, 816, 819, 823, 827, 831, 835, 839, 843, 847, 851, 855, 859, 863, 867, 871, 874, 878, 882, 886, 890, 894, 898, 901, 905, 909, 913, 917, 921, 925, 929, 933, 937, 941, 945, 949, 953, 957, 960, 963, 966, 970, 973, 976, 980, 984, 988, 992, 995, 999, 1003, 1007, 1011, 1015, 1019, 1023, 1027, 1031, 1037, 1041, 1045, 1049, 1052, 1056, 1060, 1064, 1067, 1071, 1075, 1078, 1081, 1085, 1089, 1093, 1097, 1101, 1104, 1108, 1112, 1116, 1120, 1123, 1126, 1130, 1134, 1138, 1142, 1145, 1148, 1151, 1154, 1157, 1161, 1165, 1169, 1173, 1177, 1181, 1185, 1188, 1192, 1195, 1199, 1203, 1207, 1211, 1215, 1219, 1222, 1226, 1229, 1233, 1236, 1240, 1243, 1247, 1251, 1254, 1258, 1262, 1266, 1269, 1273, 1277, 1281, 1285, 1289, 1293, 1297, 1300, 1304, 1308, 1311, 1314, 1318, 1322, 1326, 1330, 1334, 1338, 1342, 1346, 1350, 1354, 1358, 1361, 1365, 1369, 1372, 1376, 1380, 1384, 1388, 1392, 1396, 1400, 1404, 1408, 1411, 1415, 1419, 1423, 1427, 1431, 1435, 1439, 1443, 1446, 1449, 1452, 1455, 1459, 1463, 1467, 1470, 1474, 1477, 1481, 1488, 1492, 1495, 1499, 1503, 1507, 1511, 1515, 1519, 1522, 1526, 1530, 1533, 1537, 1541, 1545, 1549, 1553, 1557, 1561, 1565, 1568, 1572, 1576, 1579, 1583, 1586, 1589, 1593, 1596, 1598, 1602, 1604, 1608, 1611, 1614, 1617, 1620, 1624, 1628, 1632, 1636, 1640, 1645, 1648, 1652, 1656, 1660, 1664, 1668, 1672, 1676, 1680, 1684, 1688, 1692, 1696, 1700, 1704, 1707, 1711, 1715, 1719, 1722, 1726, 1729, 1733, 1737, 1741, 1745, 1749, 1753, 1757, 1761, 1765, 1769, 1773, 1777, 1781, 1785, 1789, 1793, 1796, 1800, 1803, 1806, 1809, 1812, 1816, 1820, 1824, 1827, 1831, 1835, 1838, 1841, 1844, 1846, 1850, 1853, 1857, 1861, 1865, 1869, 1873, 1877, 1881, 1885, 1889, 1893, 1897, 1901, 1904, 1908, 1912, 1916, 1920, 1924, 1928, 1932, 1935, 1939, 1943, 1949, 1957, 1961, 1964, 1967, 1970, 1973, 1977, 1981, 1984, 1986, 1988, 1990, 1992, 1994, 1996, 1998; and 1999-2007. Sequences that are related to the polypeptides listed in the sequence listing will have at least 46%, or at least 50%, or at least 53%, or at least 56%, or at least 61%, or at least 80%, or at least 85%, or at least 90%, or at least 100% amino acid identity to the polypeptides of the sequence listing, or comprise a conserved domain at least 80%, or at least 91%, or at least 95%, or at least 97%, or at least 100% identical to the conserved domain of a polypeptide selected from the sequence listing. The conserved domains of the polypeptides of the invention and/or found within the sequence listing are required for the function of regulating transcription and altering a trait in a transgenic plant. Transgenic plants of the invention that comprise polypeptides of the invention will have improved traits, relative to a control plant, said improved traits including larger size, larger seeds, greater yield, darker green color, increased rate of photosynthesis, more tolerance to osmotic stress, more drought tolerance, more heat tolerance, more salt tolerance, more cold tolerance, more tolerance to low nitrogen, early flowering, delayed flowering, more resistance to disease, more seed protein, and/or more seed oil relative to the control plant. The invention is also directed to methods for producing a transgenic plant, or increasing the size, yield, photosynthetic rate or yield of a plant. These methods are carried out with a target plant that is then transformed with an expression vector that encodes a polypeptide with a conserved domain at least 91%, 95%, or 97% identical to SEQ ID NO: 1995, the conserved domain of amino acids 146-194 of the G1435 polypeptide, SEQ ID NO: 1796, thus producing the transgenic plant.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING AND DRAWINGS

FIG. 1 shows a conservative estimate of phylogenetic relationships among the orders of flowering plants (modified from Angiosperm Phylogeny Group (1998) Ann. Missouri Bot. Gard. 84: 1-49). Those plants with a single cotyledon (monocots) are a monophyletic clade nested within at least two major lineages of dicots; the eudicots are further divided into rosids and asterids. Arabidopsis is a rosid eudicot classified within the order Brassicales; rice is a member of the monocot order Poales. FIG. 1 was adapted from Daly et al. (2001) Plant Physiol. 127: 1328-1333.

FIGS. 2A-2C show a Clustal W alignment of the G1435 clade. SEQ ID NOs: appear in parentheses after each Gene IDentifier (GID). The highly conserved GARP domain is identified in FIGS. 2A-2B by the box that appears around the residues within these domains.

The Sequence Listing provides exemplary polynucleotide and polypeptide sequences of the invention. CD-ROMs Copy 1 and Copy 2, and the CRF copy of the Sequence Listing under CFR Section 1.821(e), are read-only memory computer-readable compact discs. Each contains a copy of the Sequence Listing in ASCII text format. The Sequence Listing is named “MB10022-2CIP.ST25.txt”, the electronic file of the Sequence Listing contained on each of these CD-ROMs was created on Jun. 30, 2006, and is 4299 kilobytes in size. The copies of the Sequence Listing on the CD-ROM discs are hereby incorporated by reference in their entirety.

DETAILED DESCRIPTION

The present invention relates to polynucleotides and polypeptides, e.g. for modifying phenotypes of plants.

The polynucleotides of the invention encode plant transcription factors. The plant transcription factors are derived, e.g., from Arabidopsis thaliana and can belong, e.g., to one or more of the following transcription factor families: the AP2 (APETALA2) domain transcription factor family (Riechmann and Meyerowitz (1998) J. Biol. Chem. 379:633-646); the MYB transcription factor family (Martin and Paz-Ares (1997) Trends Genet. 13:67-73); the MADS domain transcription factor family (Riechmann and Meyerowitz (1997) J. Biol. Chem. 378:1079-1101); the WRKY protein family (Ishiguro and Nakamura (1994) Mol. Gen. Genet. 244:563-571); the ankyrin-repeat protein family (Zhang et al. (1992) Plant Cell 4:1575-1588); the miscellaneous protein (MISC) family (Kim et al. (1997) Plant J. 11:1237-1251); the zinc finger protein (Z) family (Klug and Schwabe (1995) FASEB J. 9: 597-604); the homeobox (HB) protein family (Duboule (1994) Guidebook to the Homeobox Genes, Oxford University Press); the CAAT-element binding proteins (Forsburg and Guarente (1989) Genes Dev. 3:1166-1178); the squamosa promoter binding proteins (SPB) (Klein et al. (1996) Mol. Gen. Genet. 1996 250:7-16); the NAM protein family; the IAA/AUX proteins (Rouse et al. (1998) Science 279:1371-1373); the HLH/MYC protein family (Littlewood et al. (1994) Prot. Profile 1:639-709); the DNA-binding protein (DBP) family (Tucker et al. (1994) EMBO J. 13:2994-3002); the bZIP family of transcription factors (Foster et al. (1994) FASEB J. 8:192-200); the BPF-1 protein (Box P-binding factor) family (da Costa e Silva et al. (1993) Plant J. 4:125-135); and the golden protein (GLD) family (Hall et al. (1998) Plant Cell 10:925-936).

In addition to methods for modifying a plant phenotype by employing one or more polynucleotides and polypeptides of the invention described herein, the polynucleotides and polypeptides of the invention have a variety of additional uses. These uses include their use in the recombinant production (i.e, expression) of proteins; as regulators of plant gene expression, as diagnostic probes for the presence of complementary or partially complementary nucleic acids (including for detection of natural coding nucleic acids); as substrates for further reactions, e.g., mutation reactions, PCR reactions, or the like, of as substrates for cloning e.g., including digestion or ligation reactions, and for identifying exogenous or endogenous modulators of the transcription factors.

Definitions

A “polynucleotide” is a nucleic acid sequence comprising a plurality of polymerized nucleotide residues, e.g., at least about 15 consecutive polymerized nucleotide residues, optionally at least about 30 consecutive nucleotides, at least about 50 consecutive nucleotides. In many instances, a polynucleotide comprises a nucleotide sequence encoding a polypeptide (or protein) or a domain or fragment thereof. Additionally, the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5′ or 3′ untranslated regions, a reporter gene, a selectable marker, or the like. The polynucleotide can be single stranded or double stranded DNA or RNA. The polynucleotide optionally comprises modified bases or a modified backbone. The polynucleotide can be, e.g., genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide can comprise a sequence in either sense or antisense orientations.

A “recombinant polynucleotide” is a polynucleotide that is not in its native state, e.g., the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context other than that in which it is naturally found, e.g., separated from nucleotide sequences with which it typically is in proximity in nature, or adjacent (or contiguous with) nucleotide sequences with which it typically is not in proximity. For example, the sequence at issue can be cloned into a vector, or otherwise recombined with one or more additional nucleic acid.

An “isolated polynucleotide” is a polynucleotide whether naturally occurring or recombinant, that is present outside the cell in which it is typically found in nature, whether purified or not. Optionally, an isolated polynucleotide is subject to one or more enrichment or purification procedures, e.g., cell lysis, extraction, centrifugation, precipitation, or the like.

A “recombinant polypeptide” is a polypeptide produced by translation of a recombinant polynucleotide. An “isolated polypeptide,” whether a naturally occurring or a recombinant polypeptide, is more enriched in (or out of) a cell than the polypeptide in its natural state in a wild type cell, e.g., more than about 5% enriched, more than about 10% enriched, or more than about 20%, or more than about 50%, or more, enriched, i.e., alternatively denoted: 105%, 110%, 120%, 150% or more, enriched relative to wild type or other control standardized at 100%. Such an enrichment is not the result of a natural response of a wild type or other control plant. Alternatively, or additionally, the isolated polypeptide is separated from other cellular components with which it is typically associated, e.g., by any of the various protein purification methods herein.

The term “plant” includes whole plants, shoot vegetative organs/structures (for example, leaves, stems and tubers), roots, flowers and floral organs/structures (for example, bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (for example, vascular tissue, ground tissue, and the like) and cells (for example, guard cells, egg cells, and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, and multicellular algae (see for example, Daly et al. (2001) Plant Physiol. 127: 1328-1333 (2001), adapted from Ku et al. (2000) Proc. Natl. Acad. Sci. USA 97: 9121-9126; and see also Tudge (2000) in The Variety of Life, Oxford University Press, New York, N.Y. pp. 547-606.

The term “transgenic plant” refers to a plant that contains genetic material, not found in a control plant such as a wild type plant of the same species, variety or cultivar. The genetic material may include a transgene, an insertional mutagenesis event (such as by transposon or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, a homologous recombination event or a sequence modified by chimeraplasty. Typically, the foreign genetic material has been introduced into the plant by human manipulation.

A transgenic plant may contain an expression vector or cassette. The expression cassette typically comprises a polypeptide-encoding sequence operably linked (i.e., under regulatory control of) to appropriate inducible or constitutive regulatory sequences that allow for the expression of polypeptide. The expression cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant. A plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, e.g., a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.

The phrase “ectopically expression or altered expression” in reference to a polynucleotide indicates that the pattern of expression in, e.g., a transgenic plant or plant tissue, is different from the expression pattern in a wild type plant, control plant, or a reference plant of the same species. For example, the polynucleotide or polypeptide is expressed in a cell or tissue type other than a cell or tissue type in which the sequence is expressed in the control or wild type plant, or by expression at a time other than at the time the sequence is expressed in the control or wild type plant, or by a response to different inducible agents, such as hormones or environmental signals, or at different expression levels (either higher or lower) compared with those found in a control or wild type plant. The term also refers to altered expression patterns that are produced by lowering the levels of expression to below the detection level or completely abolishing expression. The resulting expression pattern can be transient or stable, constitutive or inducible. In reference to a polypeptide, the term “ectopic expression or altered expression” further may relate to altered activity levels resulting from the interactions of the polypeptides with exogenous or endogenous modulators or from interactions with factors or as a result of the chemical modification of the polypeptides.

The term “fragment” or “domain,” with respect to a polypeptide, refers to a subsequence of the polypeptide. In some cases, the fragment or domain, is a subsequence of the polypeptide which performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA binding domain that binds to a DNA promoter region, an activation domain or a domain for protein-protein interactions. Fragments can vary in size from as few as 6 amino acids to the full length of the intact polypeptide, but are preferably at least about 30 amino acids in length and more preferably at least about 60 amino acids in length. In reference to a nucleotide sequence, “a fragment” refers to any subsequence of a polynucleotide, typically, of at least consecutive about 15 nucleotides, preferably at least about 30 nucleotides, more preferably at least about 50, of any of the sequences provided herein.

A “conserved domain” or “conserved region” as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences. A “GARP” domain”, such as is found in a polypeptide member of GARP family, is an example of a conserved domain. With respect to polynucleotides encoding presently disclosed polypeptides, a conserved domain is preferably at least nine base pairs (bp) in length. A conserved domain with respect to presently disclosed polypeptides refers to a domain within a polypeptide family that exhibits a higher degree of sequence homology, such as at least about 80% sequence identity, or at least about 91% sequence identity, or at least about 95% sequence identity, or at least about 97% amino acid residue sequence identity, to a conserved domain of a polypeptide of the invention (e.g., SEQ ID NOs: 1999-2007). Sequences that possess or encode for conserved domains that meet these criteria of percentage identity, and that have comparable biological activity to the present polypeptide sequences, for example, those sequences that are members of the G1435 clade polypeptides, are encompassed by the invention. A fragment or domain can be referred to as outside a conserved domain, outside a consensus sequence, or outside a consensus DNA-binding site that is known to exist or that exists for a particular polypeptide class, family, or sub-family. In this case, the fragment or domain will not include the exact amino acids of a consensus sequence or consensus DNA-binding site of a transcription factor class, family or sub-family, or the exact amino acids of a particular transcription factor consensus sequence or consensus DNA-binding site. Furthermore, a particular fragment, region, or domain of a polypeptide, or a polynucleotide encoding a polypeptide, can be “outside a conserved domain” if all the amino acids of the fragment, region, or domain fall outside of a defined conserved domain(s) for a polypeptide or protein. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.

As one of ordinary skill in the art recognizes, conserved domains may be identified as regions or domains of identity to a specific consensus sequence (see, for example, Riechmann et al. (2000a) Science 290, 2105-2110; Riechmann, J. L., and Ratcliffe, O. J. (2000b) Curr. Opin. Plant Biol. 3, 423-434). Thus, by using alignment methods well known in the art, the conserved domains of the plant polypeptides, for example, for the GARP family of transcription factors), may be determined.

The polypeptide may comprise 1) a localization domain, 2) an activation domain, 3) a repression domain, 4) an oligomerization domain, or 5) a DNA-binding domain, or the like. The conserved domains of the polypeptides of the invention and/or that are found within the sequence listing are required for the function of regulating transcription and altering a trait in a transgenic plant of the invention. Altered traits that may be conferred to a transgenic plant of the invention may include larger size, larger seeds, greater yield, darker green, increased rate of photosynthesis, more tolerance to osmotic stress, more drought tolerance, more heat tolerance, more salt tolerance, more cold tolerance, more tolerance to low nitrogen, early flowering, delayed flowering, more resistance to disease, more seed protein, and more seed oil relative to a control plant.

Conserved domains for some examples of polypeptide sequences of the invention are listed in Table 4. Also, the polypeptides of Table 4 have conserved domains specifically indicated by amino acid coordinate start and stop sites. A comparison of the regions of these polypeptides allows one of skill in the art (see, for example, Reeves and Nissen (1990) J. Biol. Chem. 265, 8573-8582) to identify domains or conserved domains for any of the polypeptides listed or referred to in this disclosure.

The term “trait” refers to a physiological, morphological, biochemical or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by available biochemical techniques, such as the protein, starch or oil content of seed or leaves or by the observation of the expression level of genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays or reporter gene expression systems, or by agricultural observations such as stress tolerance, yield or pathogen tolerance.

“Trait modification” refers to a detectable difference in a characteristic in a plant ectopically expressing a polynucleotide or polypeptide of the present invention relative to a plant not doing so, such as a control or wild type plant. In some cases, the trait modification can be evaluated quantitatively. For example, the trait modification can entail at least about a 2% increase or decrease in an observed trait (difference), at least a 5% difference, at least about a 10% difference, at least about a 20% difference, at least about a 30%, at least about a 50%, at least about a 70%, or at least about a 100%, or an even greater difference. It is known that there can be a natural variation in the modified trait. Therefore, the trait modification observed entails a change of the normal distribution of the trait in the plants compared with the distribution observed in a control or a wild type plant.

Trait modifications of particular interest include those to seed (such as embryo or endosperm), fruit, root, flower, leaf, stem, shoot, seedling or the like, including: enhanced tolerance to environmental conditions including freezing, chilling, heat, drought, water saturation, radiation and ozone; improved tolerance to microbial, fungal or viral diseases; improved tolerance to pest infestations, including nematodes, mollicutes, parasitic higher plants or the like; decreased herbicide sensitivity; improved tolerance of heavy metals or enhanced ability to take up heavy metals; improved growth under poor photoconditions (e.g., low light and/or short day length), or changes in expression levels of genes of interest. Other phenotype that can be modified relate to the production of plant metabolites, such as variations in the production of taxol, tocopherol, tocotrienol, sterols, phytosterols, vitamins, wax monomers, anti-oxidants, amino acids, lignins, cellulose, tannins, prenyllipids (such as chlorophylls and carotenoids), glucosinolates, and terpenoids, enhanced or compositionally altered protein or oil production (especially in seeds), or modified sugar (insoluble or soluble) and/or starch composition. Physical plant characteristics that can be modified include cell development (such as the number of trichomes), fruit and seed size and number, yields of plant parts such as stems, leaves and roots, the stability of the seeds during storage, characteristics of the seed pod (e.g., susceptibility to shattering), root hair length and quantity, internode distances, or the quality of seed coat. Plant growth characteristics that can be modified include growth rate, germination rate of seeds, vigor of plants and seedlings, leaf and flower senescence, male sterility, apomixis, flowering time, flower abscission, rate of nitrogen uptake, biomass or transpiration characteristics, as well as plant architecture characteristics such as apical dominance, branching patterns, number of organs, organ identity, organ shape or size.

A “control plant” as used in the present invention refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant used to compare against transgenic or genetically modified plant for the purpose of identifying an enhanced phenotype in the transgenic or genetically modified plant. A control plant may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant polynucleotide of the present invention that is expressed in the transgenic or genetically modified plant being evaluated. In general, a control plant is a plant of the same line or variety as the transgenic or genetically modified plant being tested. A suitable control plant would include a genetically unaltered or non-transgenic plant of the parental line used to generate a transgenic plant herein.

Polypeptides and Polynucleotides of the Invention

The present invention provides, among other things, transcription factors (TFs), and transcription factor homologue polypeptides, and isolated or recombinant polynucleotides encoding the polypeptides. These polypeptides and polynucleotides may be employed to modify a plant's characteristic.

Exemplary polynucleotides encoding the polypeptides of the invention were identified in the Arabidopsis thaliana GenBank database using publicly available sequence analysis programs and parameters. Sequences initially identified were then further characterized to identify sequences comprising specified sequence strings corresponding to sequence motifs present in families of known transcription factors. Polynucleotide sequences meeting such criteria were confirmed as transcription factors.

Additional polynucleotides of the invention were identified by screening Arabidopsis thaliana and/or other plant cDNA libraries with probes corresponding to known transcription factors under low stringency hybridization conditions. Additional sequences, including full length coding sequences were subsequently recovered by the rapid amplification of cDNA ends (RACE) procedure, using a commercially available kit according to the manufacturer's instructions. Where necessary, multiple rounds of RACE are performed to isolate 5′ and 3′ ends. The full length cDNA was then recovered by a routine end-to-end polymerase chain reaction (PCR) using primers specific to the isolated 5′ and 3′ ends. Exemplary sequences are provided in the Sequence Listing.

The polynucleotides of the invention were ectopically expressed in overexpressor or knockout plants and changes in the characteristics of the plants were observed. Therefore, the polynucleotides and polypeptides can be employed to improve the characteristics of plants.

Making Polynucleotides

The polynucleotides of the invention include sequences that encode transcription factors and transcription factor homologue polypeptides and sequences complementary thereto, as well as unique fragments of coding sequence, or sequence complementary thereto. Such polynucleotides can be, e.g., DNA or RNA, e.g., mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA, oligonucleotides, etc. The polynucleotides are either double-stranded or single-stranded, and include either, or both sense (i.e., coding) sequences and antisense (i.e., non-coding, complementary) sequences. The polynucleotides include the coding sequence of a transcription factor, or transcription factor homologue polypeptide, in isolation, in combination with additional coding sequences (e.g., a purification tag, a localization signal, as a fusion-protein, as a pre-protein, or the like), in combination with non-coding sequences (e.g., introns or inteins, regulatory elements such as promoters, enhancers, terminators, and the like), and/or in a vector or host environment in which the polynucleotide encoding a transcription factor or transcription factor homologue polypeptide is an endogenous or exogenous gene.

A variety of methods exist for producing the polynucleotides of the invention. Procedures for identifying and isolating DNA clones are well known to those of skill in the art, and are described in, e.g., Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (“Berger”); Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2000) (“Ausubel”).

Alternatively, polynucleotides of the invention, can be produced by a variety of in vitro amplification methods adapted to the present invention by appropriate selection of specific or degenerate primers. Examples of protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Qbeta-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA), e.g., for the production of the homologous nucleic acids of the invention are found in Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987) PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990) (Innis). Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369: 684-685 and the references cited therein, in which PCR amplicons of up to 40 kb are generated. One of skill will appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, e.g., Ausubel, Sambrook and Berger, all supra.

Alternatively, polynucleotides and oligonucleotides of the invention can be assembled from fragments produced by solid-phase synthesis methods. Typically, fragments of up to approximately 100 bases are individually synthesized and then enzymatically or chemically ligated to produce a desired sequence, e.g., a polynucleotide encoding all or part of a transcription factor. For example, chemical synthesis using the phosphoramidite method is described, e.g., by Beaucage et al. (1981) Tetrahedron Letters 22:1859-69; and Matthes et al. (1984) EMBO J. 3:801-5. According to such methods, oligonucleotides are synthesized, purified, annealed to their complementary strand, ligated and then optionally cloned into suitable vectors. And if so desired, the polynucleotides and polypeptides of the invention can be custom ordered from any of a number of commercial suppliers.

Table 1 provides exemplary polynucleotide sequences of the invention. The table includes from left to right for each sequence: the SEQ ID No., the internal code reference number (GID), where the coding sequence is initiated and where the coding sequence terminates, and identification of any conserved domains for the translated polypeptide sequences maintaining the same coordinates of the polynucleotide sequence.

TABLE 1 Exemplary Polynucleotide Sequences of the Invention Coding Sequence Conserved Domain(s) in SEQ ID No. GID Coordinates Amino Acid Coordinates 1 G188 50 . . . 1096 175-222 2 G616 129 . . . 1211 39-95 3 G19 70 . . . 816  76-145 4 G261 458 . . . 1663  16-104 5 G28 63 . . . 869 145-213 6 G869 428 . . . 1402 109-177 7 G237 1 . . . 852  11-113 8 G409 331 . . . 1149  64-124 9 G418 103 . . . 2322 500-560 10 G591 88 . . . 1020 143-240 11 G525 109 . . . 966  23-167 12 G545 55 . . . 738 82-102, 136-154 13 G865 282 . . . 920  36-103 14 G881 76 . . . 1008 176-233 15 G896 47 . . . 1150 18-39 16 G378 1 . . . 726 196-237 17 G569 184 . . . 969  90-153 18 G558 267 . . . 1259  45-105 19 G22 81 . . . 761  89-157 20 G225 157 . . . 441 39-76 21 G226 10 . . . 348 28-78 22 G256 312 . . . 1310  13-115 23 G419 381 . . . 2213 392-452 24 G464 41 . . . 664 7-15, 70-80, 125-158, 183-219 25 G482 188 . . . 760  25-116 26 G502 224 . . . 1093  10-155 27 G526 181 . . . 1188  21-149 28 G561 86 . . . 1168 248-308 29 G664 104 . . . 952  13-116 30 G682 1 . . . 228 22-53 31 G911 1 . . . 480  86-129 32 G964 162 . . . 1013 126-186 33 G394 82 . . . 918 121-182 34 G489 33 . . . 695  57-156 35 G214 238 . . . 2064 22-71 36 G229 41 . . . 1156  14-120 37 G241 46 . . . 867  14-114 38 G663 113 . . . 862  9-111 39 G776 76 . . . 1431  27-175 40 G778 50 . . . 1249 220-267 41 G883 67 . . . 1041 245-302 42 G938 1 . . . 1755  96-104 43 G1328 67 . . . 1041  14-119 44 G584 40 . . . 1809 401-494 45 G668 1 . . . 1056  13-113 46 G727 43 . . . 1977 226-269 47 G732 73 . . . 588 31-91 48 G9 81 . . . 1139  62-127 49 G428 97 . . . 1032 229-292 50 G1269 88 . . . 951 27-83 51 G1038 240 . . . 1574 198-247 52 G438 188 . . . 2716 22-85 53 G571 326 . . . 1708 160-220 54 G748 98 . . . 1444 112-140 55 G431 1 . . . 1149 286-335 56 G187 118 . . . 1074 172-228 57 G470 1 . . . 2580  61-393 58 G615 197 . . . 1252  88-147 59 G1073 62 . . . 874 33-42, 78-175 60 G26 73 . . . 729  67-134 61 G38 149 . . . 1156  76-143 62 G43 38 . . . 643 104-172 63 G207 16 . . . 930  6-106 64 G254 15 . . . 923  62-106 65 G263 48 . . . 902  15-105 66 G308 196 . . . 1794 270-274 67 G536 1 . . . 768 226-233 68 G680 338 . . . 2275 24-70 69 G867 64 . . . 1098  59-124 70 G912 20 . . . 694  51-118 71 G996 53 . . . 1063  14-114 72 G1068 150 . . . 1310 143-150 73 G1337 97 . . . 1398  9-75 74 G231 88 . . . 888  14-118 75 G274 172 . . . 2037 108-572 76 G307 1 . . . 1764 323-339 77 G346 1 . . . 825 196-221 78 G598 248 . . . 1039 205-263 79 G605 72 . . . 1076 132-143 80 G777 54 . . . 914  47-101 81 G1133 104 . . . 1084 256-326 82 G1266 62 . . . 718  79-147 83 G1324 54 . . . 914  20-118 84 G975 58 . . . 657  4-71 85 G157 31 . . . 621  2-57 86 G859 132 . . . 569  2-57 87 G1842 219 . . . 809  2-57 88 G1843 51 . . . 653  2-57 89 G1844 39 . . . 635  2-57 90 G861 158 . . . 880  2-57 91 G192 63 . . . 959 128-185 92 G234 106 . . . 1035  14-115 93 G361 54 . . . 647 43-63 94 G486 1 . . . 420  5-66 95 G994 180 . . . 917  14-123 96 G1335 56 . . . 667 24-43, 131-144, 185-203 97 G562 137 . . . 1285 253-315 98 G736 1 . . . 513  54-111 99 G1435 8 . . . 904 146-194 100 G180 54 . . . 629 118-174 101 G592 121 . . . 1200 290-342 102 G208 15 . . . 725  14-116 103 G658 17 . . . 757  2-105 104 G1334 76 . . . 885  18-190 105 G27 83 . . . 622  37-104 106 G740 25 . . . 924 24-42, 232-268 107 G559 89 . . . 1285 203-264 108 G1093 1 . . . 531 105-148 109 G725 46 . . . 1122 39-87 Orthologs and Paralogs

Sequences homologous, i.e., that share significant sequence identity or similarity, to those provided in the Sequence Listing, derived from Arabidopsis thaliana or from other plants of choice are also an aspect of the invention. Homologous sequences can be derived from any plant including monocots and dicots and in particular agriculturally important plant species, including but not limited to, crops such as soybean, wheat, corn, potato, cotton, rice, oilseed rape (including canola), sunflower, alfalfa, sugarcane and turf; or fruits and vegetables, such as banana, blackberry, blueberry, strawberry, and raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, spinach, squash, sweet corn, tobacco, tomato, watermelon, rosaceous fruits (such as apple, peach, pear, cherry and plum) and vegetable brassicas (such as broccoli, cabbage, cauliflower, Brussels sprouts and kohlrabi). Other crops, fruits and vegetables whose phenotype can be changed include barley, rye, millet, sorghum, currant, avocado, citrus fruits such as oranges, lemons, grapefruit and tangerines, artichoke, cherries, nuts such as the walnut and peanut, endive, leek, roots, such as arrowroot, beet, cassava, turnip, radish, yam, and sweet potato, and beans. The homologous sequences may also be derived from woody species, such pine, poplar and eucalyptus.

Homologous sequences as described above can comprise orthologous or paralogous sequences. Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences. General methods for identifying orthologs and paralogs, including phylogenetic methods, sequence similarity and hybridization methods, are described herein; an ortholog or paralog, including equivalogs, may be identified by one or more of the methods described below.

As described by Eisen (1998) Genome Res. 8: 163-167, evolutionary information may be used to predict gene function. It is common for groups of genes that are homologous in sequence to have diverse, although usually related, functions. However, in many cases, the identification of homologs is not sufficient to make specific predictions because not all homologs have the same function. Thus, an initial analysis of functional relatedness based on sequence similarity alone may not provide one with a means to determine where similarity ends and functional relatedness begins. Fortunately, it is well known in the art that protein function can be classified using phylogenetic analysis of gene trees combined with the corresponding species. Functional predictions can be greatly improved by focusing on how the genes became similar in sequence (i.e., by evolutionary processes) rather than on the sequence similarity itself (Eisen, supra). In fact, many specific examples exist in which gene function has been shown to correlate well with gene phylogeny (Eisen, supra). Thus, “[t]he first step in making functional predictions is the generation of a phylogenetic tree representing the evolutionary history of the gene of interest and its homologs. Such trees are distinct from clusters and other means of characterizing sequence similarity because they are inferred by techniques that help convert patterns of similarity into evolutionary relationships . . . . After the gene tree is inferred, biologically determined functions of the various homologs are overlaid onto the tree. Finally, the structure of the tree and the relative phylogenetic positions of genes of different functions are used to trace the history of functional changes, which is then used to predict functions of [as yet] uncharacterized genes” (Eisen, supra).

Within a single plant species, gene duplication may cause two copies of a particular gene, giving rise to two or more genes with similar sequence and often similar function known as paralogs. A paralog is therefore a similar gene formed by duplication within the same species. Paralogs typically cluster together or in the same clade (a group of similar genes) when a gene family phylogeny is analyzed using programs such as CLUSTAL Thompson et al. (1994) Nucleic Acids Res. 22: 4673-4680; et al. (1996) Methods Enzymol. 266: 383-402). Groups of similar genes can also be identified with pair-wise BLAST analysis (Feng and Doolittle (1987) J. Mol. Evol. 25: 351-360). For example, a clade of very similar MADS domain transcription factors from Arabidopsis all share a common function in flowering time (Ratcliffe et al. (2001) Plant Physiol. 126: 122-132), and a group of very similar AP2 domain transcription factors from Arabidopsis are involved in tolerance of plants to freezing (Gilmour et al. (1998) Plant J. 16: 433-442). Analysis of groups of similar genes with similar function that fall within one clade can yield sub-sequences that are particular to the clade. These sub-sequences, known as consensus sequences, can not only be used to define the sequences within each lade, but define the functions of these genes; genes within a clade may contain paralogous sequences, or orthologous sequences that share the same function (see also, for example, Mount (2001), in Bioinformatics: Sequence and Genome Analysis, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., p. 543).

Transcription factor gene sequences are conserved across diverse eukaryotic species lines (Goodrich et al. (1993) Cell 75: 519-530); Lin et al. (1991) Nature 353: 569-571; Sadowski et al. (1988) Nature 335: 563-564). Plants are no exception to this observation; diverse plant species possess transcription factors that have similar sequences and functions. Speciation, the production of new species from a parental species, gives rise to two or more genes with similar sequence and similar function. These genes, termed orthologs, often have an identical function within their host plants and are often interchangeable between species without losing function. Because plants have common ancestors, many genes in any plant species will have a corresponding orthologous gene in another plant species. Once a phylogenic tree for a gene family of one species has been constructed using a program such as CLUSTAL (Thompson et al. (1994) supra; Higgins et al. (1996) Methods Enzymol. 266: 383-402) potential orthologous sequences can be placed into the phylogenetic tree and their relationship to genes from the species of interest can be determined. Orthologous sequences can also be identified by a reciprocal BLAST strategy. Once an orthologous sequence has been identified, the function of the ortholog can be deduced from the identified function of the reference sequence.

By using a phylogenetic analysis, one skilled in the art would recognize that the ability to predict similar functions conferred by closely-related polypeptides is predictable. This predictability has been confirmed by our own many studies in which we have found that a wide variety of polypeptides have orthologous or closely-related homologous sequences that function as does the first, closely-related reference sequence. For example, distinct transcription factors, including:

(i) AP2 family Arabidopsis G47 (found in US patent publication 20040019925A1), a phylogenetically-related sequence from soybean, and two phylogenetically-related homologs from rice all can confer greater tolerance to drought, hyperosmotic stress, or delayed flowering as compared to control plants;

(ii) CAAT family Arabidopsis G481 (found in PCT patent publication WO2004076638), and numerous phylogenetically-related sequences from dicots and monocots can confer greater tolerance to drought-related stress as compared to control plants;

(iii) Myb-related Arabidopsis G682 (found in PCT patent publication WO2004076638) and numerous phylogenetically-related sequences from dicots and monocots can confer greater tolerance to heat, drought-related stress, cold, and salt as compared to control plants;

(iv) WRKY family Arabidopsis G1274 (found in U.S. patent application Ser. No. 10/666,642) and numerous closely-related sequences from dicots and monocots have been shown to confer increased water deprivation tolerance, and

(v) AT-hook family soy sequence G3456 (found in US patent publication 20040128712A1) and numerous phylogenetically-related sequences from dicots and monocots, increased biomass compared to control plants when these sequences are overexpressed in plants.

The polypeptides sequences belong to distinct clades of polypeptides that include members from diverse species. In each case, most or all of the clade member sequences derived from both dicots and monocots have been shown to confer darker green coloration, increased photosynthetic rate, increased size, increased yield or delayed flowering, relative to control plants, when the sequences were overexpressed. These studies each demonstrate that evolutionarily conserved genes from diverse species are likely to function similarly (i.e., by regulating similar target sequences and controlling the same traits), and that polynucleotides from one species may be transformed into closely-related or distantly-related plant species to confer or improve traits.

As shown in Table 4, polypeptides that are phylogenetically related to the polypeptides of the invention may have at least 46%, 50%, 53%, 56%, 61% or 100% amino acid sequence identity with a member of the G1435 clade of transcription factors, or have conserved GARP domains that share at least 91%, 95%, 97%, or 100% amino acid sequence identity with a member of the G1435 clade of transcription factors, and have similar functions in that the polypeptides of the invention may, when overexpressed, confer at least one regulatory activity selected from the group consisting of greater increased photosynthetic rate, increased size, increased yield or delayed flowering, relative to control plants.

At the nucleotide level, the sequences of the invention will typically share at least about 84%, 85%, 87%, 89%, 90% or 100% sequence identity to one or more of the listed full-length sequences. These percentages were determined by BLASTn analysis comparing to the G1435 polynucleotide, SEQ ID NO: 99, the clade member nucleotide sequences of:

-   -   G4241 or G4240 (SEQ ID NOs: 1991 or 1993, each 84% identical to         G1435, the BLASTn analysis comparing 103/122 bases of either         sequence to G1435);     -   G4244 (SEQ ID NO: 1987, 85% identical to G1435, the BLASTn         analysis comparing 89/105 bases to G1435);     -   G4243 (SEQ ID NO: 1985, 87% identical to G1435, the BLASTn         analysis comparing 91/104 bases to G1435);     -   G4245 (SEQ ID NO: 1989, 89% identical to G1435, the BLASTn         analysis comparing 35/39 bases to G1435); or     -   G2741 (SEQ ID NO: 1983, 90% identical to G1435, the BLASTn         analysis comparing 172/191 bases to G1435).

The degeneracy of the genetic code enables major variations in the nucleotide sequence of a polynucleotide while maintaining the amino acid sequence of the encoded protein.

Percentage identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Inc. Madison, Wis.). The MEGALIGN program can create alignments between two or more sequences according to different methods, for example, the clustal method (see, for example, Higgins and Sharp (1988) Gene 73: 237-244. The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. Other alignment algorithms or programs may be used, including FASTA, BLAST, or ENTREZ, FASTA and BLAST, and which may be used to calculate percent similarity. These are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with or without default settings. ENTREZ is available through the National Center for Biotechnology Information. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences (see U.S. Pat. No. 6,262,333).

Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology Information (see internet website at http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul (1990) J. Mol. Biol. 215: 403-410; Altschul (1993) J. Mol. Evol. 36: 290-300). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915). Unless otherwise indicated for comparisons of predicted polynucleotides, “sequence identity” refers to the % sequence identity generated from a tblastx using the NCBI version of the algorithm at the default settings using gapped alignments with the filter “off” (see, for example, internet website at http://www.ncbi.nlm.nih.gov/).

Other techniques for alignment are described by Doolittle, ed. (1996) Methods in Enzymology, vol. 266: “Computer Methods for Macromolecular Sequence Analysis” Academic Press, Inc., San Diego, Calif., USA. Preferably, an alignment program that permits gaps in the sequence is utilized to align the sequences. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments (see Shpaer (1997) Methods Mol. Biol. 70: 173-187). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a Smith-Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.

The percentage similarity between two polypeptide sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between polynucleotide sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method (see, for example, Hein (1990) Methods Enzymol. 183: 626-645) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions (see US Patent Application No. 20010010913).

Thus, the invention provides methods for identifying a sequence similar or paralogous or orthologous or homologous to one or more polynucleotides as noted herein, or one or more target polypeptides encoded by the polynucleotides, or otherwise noted herein and may include linking or associating a given plant phenotype or gene function with a sequence. In the methods, a sequence database is provided (locally or across an internet or intranet) and a query is made against the sequence database using the relevant sequences herein and associated plant phenotypes or gene functions.

In addition, one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to search against a BLOCKS (Bairoch et al. (1997) Nucleic Acids Res. 25: 217-221), PFAM, and other databases which contain previously identified and annotated motifs, sequences and gene functions. Methods that search for primary sequence patterns with secondary structure gap penalties (Smith et al. (1992) Protein Engineering 5: 35-51) as well as algorithms such as Basic Local Alignment Search Tool (BLAST; Altschul (1990) supra; Altschul et al. (1993) supra), BLOCKS (Henikoff and Henikoff (1991) Nucleic Acids Res. 19: 6565-6572), Hidden Markov Models (HMM; Eddy (1996) Curr. Opin. Str. Biol. 6: 361-365; Sonnhammer et al. (1997) Proteins 28: 405-420), and the like, can be used to manipulate and analyze polynucleotide and polypeptide sequences encoded by polynucleotides. These databases, algorithms and other methods are well known in the art and are described in Ausubel et al. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., unit 7.7, and in Meyers (1995) Molecular Biology and Biotechnology, Wiley VCH, New York, N.Y., p 856-853.

A further method for identifying or confirming that specific homologous sequences control the same function is by comparison of the transcript profile(s) obtained upon overexpression or knockout of two or more related polypeptides. Since transcript profiles are diagnostic for specific cellular states, one skilled in the art will appreciate that genes that have a highly similar transcript profile (e.g., with greater than 50% regulated transcripts in common, or with greater than 70% regulated transcripts in common, or with greater than 90% regulated transcripts in common) will have highly similar functions. Fowler and Thomashow (2002) Plant Cell 14: 1675-1690), have shown that three paralogous AP2 family genes (CBF1, CBF2 and CBF3) are induced upon cold treatment, and each of which can condition improved freezing tolerance, and all have highly similar transcript profiles. Once a polypeptide has been shown to provide a specific function, its transcript profile becomes a diagnostic tool to determine whether paralogs or orthologs have the same function.

Furthermore, methods using manual alignment of sequences similar or homologous to one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to identify regions of similarity and B-box zinc finger domains. Such manual methods are well-known of those of skill in the art and can include, for example, comparisons of tertiary structure between a polypeptide sequence encoded by a polynucleotide that comprises a known function and a polypeptide sequence encoded by a polynucleotide sequence that has a function not yet determined. Such examples of tertiary structure may comprise predicted α helices, β-sheets, amphipathic helices, leucine zipper motifs, zinc finger motifs, proline-rich regions, cysteine repeat motifs, and the like.

Orthologs and paralogs of presently disclosed polypeptides may be cloned using compositions provided by the present invention according to methods well known in the art. cDNAs can be cloned using mRNA from a plant cell or tissue that expresses one of the present sequences. Appropriate mRNA sources may be identified by interrogating Northern blots with probes designed from the present sequences, after which a library is prepared from the mRNA obtained from a positive cell or tissue. Polypeptide-encoding cDNA is then isolated using, for example, PCR, using primers designed from a presently disclosed gene sequence, or by probing with a partial or complete cDNA or with one or more sets of degenerate probes based on the disclosed sequences. The cDNA library may be used to transform plant cells. Expression of the cDNAs of interest is detected using, for example, microarrays, Northern blots, quantitative PCR, or any other technique for monitoring changes in expression. Genomic clones may be isolated using similar techniques to those.

Examples of orthologs of Arabidopsis polypeptide sequences and their functionally similar orthologs are listed in Table 4 and the Sequence Listing. In addition to the sequences in Table 4 and the Sequence Listing, the invention encompasses isolated nucleotide sequences that are phylogenetically and structurally similar to sequences listed in the Sequence Listing) and can function in a plant by increasing yield and/or and abiotic stress tolerance when ectopically expressed in a plant.

Since a significant number of these sequences are phylogenetically and sequentially related to each other and have been shown to increase yield from a plant and/or abiotic stress tolerance, one skilled in the art would predict that other similar, phylogenetically related sequences falling within the present clades of polypeptides would also perform similar functions when ectopically expressed.

Identifying Nucleic Acids by Hybridization

Polynucleotides homologous to the sequences illustrated in the Sequence Listing can be identified, e.g., by hybridization to each other under stringent or under highly stringent conditions. Single stranded polynucleotides hybridize when they associate based on a variety of well characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. The stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency is influenced by a variety of factors, including temperature, salt concentration and composition, organic and non-organic additives, solvents, etc. present in both the hybridization and wash solutions and incubations (and number), as described in more detail in the references cited above.

An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is about 5° C. to 20° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The T_(m) is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Nucleic acid molecules that hybridize under stringent conditions will typically hybridize to a probe based on either the entire cDNA or selected portions, e.g., to a unique subsequence, of the cDNA under wash conditions of 0.2×SSC to 2.0×SSC, 0.1% SDS at 50-65° C., for example 0.2×SSC, 0.1% SDS at 65° C. For identification of less closely related homologues washes can be performed at a lower temperature, e.g., 50° C. In general, stringency is increased by raising the wash temperature and/or decreasing the concentration of SSC.

As another example, stringent conditions can be selected such that an oligonucleotide that is perfectly complementary to the coding oligonucleotide hybridizes to the coding oligonucleotide with at least about a 5-10× higher signal to noise ratio than the ratio for hybridization of the perfectly complementary oligonucleotide to a nucleic acid encoding a transcription factor known as of the filing date of the application. Conditions can be selected such that a higher signal to noise ratio is observed in the particular assay which is used, e.g., about 15×, 25×, 35×, 50× or more. Accordingly, the subject nucleic acid hybridizes to the unique coding oligonucleotide with at least a 2× higher signal to noise ratio as compared to hybridization of the coding oligonucleotide to a nucleic acid encoding known polypeptide. Again, higher signal to noise ratios can be selected, e.g., about 5×, 10×, 25×, 35×, 50× or more. The particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a calorimetric label, a radioactive label, or the like.

Alternatively, transcription factor homologue polypeptides can be obtained by screening an expression library using antibodies specific for one or more transcription factors. With the provision herein of the disclosed transcription factor, and transcription factor homologue nucleic acid sequences, the encoded polypeptide(s) can be expressed and purified in a heterologous expression system (e.g., E. coli) and used to raise antibodies (monoclonal or polyclonal) specific for the polypeptide(s) in question. Antibodies can also be raised against synthetic peptides derived from transcription factor, or transcription factor homologue, amino acid sequences. Methods of raising antibodies are well known in the art and are described in Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Such antibodies can then be used to screen an expression library produced from the plant from which it is desired to clone additional transcription factor homologues, using the methods described above. The selected cDNAs can be confirmed by sequencing and enzymatic activity.

Sequence Variations

It will readily be appreciated by those of skill in the art, that any of a variety of polynucleotide sequences are capable of encoding the transcription factors and transcription factor homologue polypeptides of the invention. Due to the degeneracy of the genetic code, many different polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing.

For example, Table 2 illustrates, e.g., that the codons AGC, AGT, TCA, TCC, TCG, and TCT all encode the same amino acid: serine. Accordingly, at each position in the sequence where there is a codon encoding serine, any of the above trinucleotide sequences can be used without altering the encoded polypeptide.

TABLE 2 Amino acids Codon Alanine Ala A GCA GCC GCG GCU Cysteine Cys C TGC TGT Aspartic acid Asp D GAC GAT Glutamic acid Glu E GAA GAG Phenylalanine Phe F TTC TTT Glycine Gly G GGA GGC GGG GGT Histidine His H CAC CAT Isoleucine Ile I ATA ATC ATT Lysine Lys K AAA AAG Leucine Leu L TTA TTG CTA CTC CTG CTT Methionine Met M ATG Asparagine Asn N AAC AAT Proline Pro P CCA CCC CCG CCT Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGT Serine Ser S AGG AGT TCA TCC TCG TCT Threonine Thr T ACA ACC ACG ACT Valine Val V GTA GTC GTG GTG GTT Tryptophan Trp W TGG Tyrosine Tyr Y TAC TAT

Sequence alterations that do not change the amino acid sequence encoded by the polynucleotide are termed “silent” variations. With the exception of the codons ATG and TGG, encoding methionine and tryptophan, respectively, any of the possible codons for the same amino acid can be substituted by a variety of techniques, e.g., site-directed mutagenesis, available in the art. Accordingly, any and all such variations of a sequence selected from the above table are a feature of the invention.

In addition to silent variations, other conservative variations that alter one, or a few amino acids in the encoded polypeptide, can be made without altering the function of the polypeptide, these conservative variants are, likewise, a feature of the invention.

For example, substitutions, deletions and insertions introduced into the sequences provided in the Sequence Listing are also envisioned by the invention. Such sequence modifications can be engineered into a sequence by site-directed mutagenesis (Wu (ed.) Meth. Enzymol. (1993) vol. 217, Academic Press) or the other methods noted below. Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. In preferred embodiments, deletions or insertions are made in adjacent pairs, e.g., a deletion of two residues or insertion of two residues. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a sequence. The mutations that are made in the polynucleotide encoding the transcription factor should not place the sequence out of reading frame and should not create complementary regions that could produce secondary mRNA structure. Preferably, the polypeptide encoded by the DNA performs the desired function.

Conservative substitutions are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the Table 3 when it is desired to maintain the activity of the protein. Table 3 shows amino acids which can be substituted for an amino acid in a protein and which are typically regarded as conservative substitutions.

TABLE 3 Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln; His Asp Glu Gln Asn Cys Ser Glu Asp Gly Pro His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln Met Leu; Ile Phe Met; Leu; Tyr Ser Thr; Gly Thr Ser; Val Trp Tyr Tyr Trp; Phe Val Ile; Leu

Substitutions that are less conservative than those in Table 3 can be selected by picking residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.

Further Modifying Sequences of the Invention—Mutation/Forced Evolution

In addition to generating silent or conservative substitutions as noted, above, the present invention optionally includes methods of modifying the sequences of the Sequence Listing. In the methods, nucleic acid or protein modification methods are used to alter the given sequences to produce new sequences and/or to chemically or enzymatically modify given sequences to change the properties of the nucleic acids or proteins.

Thus, in one embodiment, given nucleic acid sequences are modified, e.g., according to standard mutagenesis or artificial evolution methods to produce modified sequences. For example, Ausubel, supra, provides additional details on mutagenesis methods. Artificial forced evolution methods are described, e.g., by Stemmer (1994) Nature 370:389-391, and Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Many other mutation and evolution methods are also available and expected to be within the skill of the practitioner.

Similarly, chemical or enzymatic alteration of expressed nucleic acids and polypeptides can be performed by standard methods. For example, sequence can be modified by addition of lipids, sugars, peptides, organic or inorganic compounds, by the inclusion of modified nucleotides or amino acids, or the like. For example, protein modification techniques are illustrated in Ausubel, supra. Further details on chemical and enzymatic modifications can be found herein. These modification methods can be used to modify any given sequence, or to modify any sequence produced by the various mutation and artificial evolution modification methods noted herein.

Accordingly, the invention provides for modification of any given nucleic acid by mutation, evolution, chemical or enzymatic modification, or other available methods, as well as for the products produced by practicing such methods, e.g., using the sequences herein as a starting substrate for the various modification approaches.

For example, optimized coding sequence containing codons preferred by a particular prokaryotic or eukaryotic host can be used e.g., to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced using a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, preferred stop codons for S. cerevisiae and mammals are TAA and TGA, respectively. The preferred stop codon for monocotyledonous plants is TGA, whereas insects and E. coli prefer to use TAA as the stop codon.

The polynucleotide sequences of the present invention can also be engineered in order to alter a coding sequence for a variety of reasons, including but not limited to, alterations which modify the sequence to facilitate cloning, processing and/or expression of the gene product. For example, alterations are optionally introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, to change codon preference, to introduce splice sites, etc.

Furthermore, a fragment or domain derived from any of the polypeptides of the invention can be combined with domains derived from other transcription factors or synthetic domains to modify the biological activity of a transcription factor. For instance, a DNA binding domain derived from a transcription factor of the invention can be combined with the activation domain of another transcription factor or with a synthetic activation domain. A transcription activation domain assists in initiating transcription from a DNA binding site. Examples include the transcription activation region of VP16 or GAL4 (Moore et al. (1998) Proc. Natl. Acad. Sci. USA 95: 376-381; and Aoyama et al. (1995) Plant Cell 7:1773-1785), peptides derived from bacterial sequences (Ma and Ptashne (1987) Cell 51; 113-119) and synthetic peptides (Giniger and Ptashne, (1987) Nature 330:670-672).

Expression and Modification of Polypeptides

Typically, polynucleotide sequences of the invention are incorporated into recombinant DNA (or RNA) molecules that direct expression of polypeptides of the invention in appropriate host cells, transgenic plants, in vitro translation systems, or the like. Due to the inherent degeneracy of the genetic code, nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence can be substituted for any listed sequence to provide for cloning and expressing the relevant homologue.

Vectors, Promoters and Expression Systems

The present invention includes recombinant constructs comprising one or more of the nucleic acid sequences herein. The constructs typically comprise a vector, such as a plasmid, a cosmid, a phage, a virus (e.g., a plant virus), a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or the like, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.

General texts which describe molecular biological techniques useful herein, including the use and production of vectors, promoters and many other relevant topics, include Berger, Sambrook and Ausubel, supra. Any of the identified sequences can be incorporated into a cassette or vector, e.g., for expression in plants. A number of expression vectors suitable for stable transformation of plant cells or for the establishment of transgenic plants have been described including those described in Weissbach and Weissbach, (1989) Methods for Plant Molecular Biology, Academic Press, and Gelvin et al., (1990) Plant Molecular Biology Manual, Kluwer Academic Publishers. Specific examples include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed by Herrera-Estrella et al. (1983) Nature 303: 209, Bevan (1984) Nucl Acid Res. 12: 8711-8721, Klee (1985) Bio/Technol. 3: 637-642, for dicotyledonous plants.

Alternatively, non-Ti vectors can be used to transfer the DNA into monocotyledonous plants and cells by using free DNA delivery techniques. Such methods can involve, for example, the use of liposomes, electroporation, microprojectile bombardment, silicon carbide whiskers, and viruses. By using these methods transgenic plants such as wheat, rice (Christou (1991) Bio/Technol. 9: 957-962) and corn (Gordon-Kamm (1990) Plant Cell 2: 603-618) can be produced. An immature embryo can also be a good target tissue for monocots for direct DNA delivery techniques by using the particle gun (Weeks et al. (1993) Plant Physiol. 102: 1077-1084; Vasil (1993) Bio/Technol. 10: 667-674; Wan and Lemeaux (1994) Plant Physiol. 104: 37-48, and for Agrobacterium-mediated DNA transfer (Ishida et al. (1996) Nature Biotech. 14: 745-750).

Typically, plant transformation vectors include one or more cloned plant coding sequence (genomic or cDNA) under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant transformation vectors typically also contain a promoter (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, an RNA processing signal (such as intron splice sites), a transcription termination site, and/or a polyadenylation signal.

Examples of constitutive plant promoters which can be useful for expressing the TF sequence include: the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (see, e.g., Odel et al. (1985) Nature 313:810); the nopaline synthase promoter (An et al. (1988) Plant Physiol. 88:547); and the octopine synthase promoter (Fromm et al. (1989) Plant Cell 1: 977).

A variety of plant gene promoters that regulate gene expression in response to environmental, hormonal, chemical, developmental signals, and in a tissue-active manner can be used for expression of a TF sequence in plants. Choice of a promoter is based largely on the phenotype of interest and is determined by such factors as tissue (e.g., seed, fruit, root, pollen, vascular tissue, flower, carpel, etc.), inducibility (e.g., in response to wounding, heat, cold, drought, light, pathogens, etc.), timing, developmental stage, and the like. Numerous known promoters have been characterized and can favorable be employed to promote expression of a polynucleotide of the invention in a transgenic plant or cell of interest. For example, tissue specific promoters include: seed-specific promoters (such as the napin, phaseolin or DC3 promoter described in U.S. Pat. No. 5,773,697), fruit-specific promoters that are active during fruit ripening (such as the dru 1 promoter (U.S. Pat. No. 5,783,393), or the 2A11 promoter (U.S. Pat. No. 4,943,674) and the tomato polygalacturonase promoter (Bird et al. (1988) Plant Mol Biol. 11:651), root-specific promoters, such as those disclosed in U.S. Pat. Nos. 5,618,988, 5,837,848 and 5,905,186, pollen-active promoters such as PTA29, PTA26 and PTA13 (U.S. Pat. No. 5,792,929), promoters active in vascular tissue (Ringli and Keller (1998) Plant Mol. Biol. 37:977-988), flower-specific (Kaiser et al, (1995) Plant Mol. Biol. 28:231-243), pollen (Baerson et al. (1994) Plant Mol. Biol. 26:1947-1959), carpels (Ohl et al. (1990) Plant Cell 2:837-848), pollen and ovules (Baerson et al. (1993) Plant Mol. Biol. 22:255-267), auxin-inducible promoters (such as that described in van der Kop et al. (1999) Plant Mol. Biol. 39:979-990 or Baumann et al. (1999) Plant Cell 11:323-334), cytokinin-inducible promoter (Guevara-Garcia (1998) Plant Mol. Biol. 38:743-753), promoters responsive to gibberellin (Shi et al. (1998) Plant Mol. Biol. 38:1053-1060, Willmott et al. (1998) 38:817-825) and the like. Additional promoters are those that elicit expression in response to heat (Ainley et al. (1993) Plant Mol. Biol. 22: 13-23), light (e.g., the pea rbcS-3A promoter, Kuhlemeier et al. (1989) Plant Cell 1:471, and the maize rbcS promoter, Schaffner and Sheen (1991) Plant Cell 3: 997); wounding (e.g., wunI, Siebertz et al. (1989) Plant Cell 1: 961); pathogens (such as the PR-1 promoter described in Buchel et al. (1999) Plant Mol. Biol. 40:387-396, and the PDF1.2 promoter described in Manners et al. (1998) Plant Mol. Biol. 38:1071-80), and chemicals such as methyl jasmonate or salicylic acid (Gatz et al. (1997) Plant Mol. Biol. 48: 89-108). In addition, the timing of the expression can be controlled by using promoters such as those acting at senescence (An and Amazon (1995) Science 270: 1986-1988); or late seed development (Odell et al. (1994) Plant Physiol. 106:447-458).

Plant expression vectors can also include RNA processing signals that can be positioned within, upstream or downstream of the coding sequence. In addition, the expression vectors can include additional regulatory sequences from the 3′-untranslated region of plant genes, e.g., a 3′ terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3′ terminator regions.

Additional Expression Elements

Specific initiation signals can aid in efficient translation of coding sequences. These signals can include, e.g., the ATG initiation codon and adjacent sequences. In cases where a coding sequence, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence (e.g., a mature protein coding sequence), or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon can be separately provided. The initiation codon is provided in the correct reading frame to facilitate transcription. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers appropriate to the cell system in use.

Expression Hosts

The present invention also relates to host cells which are transduced with vectors of the invention, and the production of polypeptides of the invention (including fragments thereof) by recombinant techniques. Host cells are genetically engineered (i.e, nucleic acids are introduced, e.g., transduced, transformed or transfected) with the vectors of this invention, which may be, for example, a cloning vector or an expression vector comprising the relevant nucleic acids herein. The vector is optionally a plasmid, a viral particle, a phage, a naked nucleic acids, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the relevant gene. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, Sambrook and Ausubel.

The host cell can be a eukaryotic cell, such as a yeast cell, or a plant cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Plant protoplasts are also suitable for some applications. For example, the DNA fragments are introduced into plant tissues, cultured plant cells or plant protoplasts by standard methods including electroporation (Fromm et al., (1985) Proc. Natl. Acad. Sci. USA 82, 5824, infection by viral vectors such as cauliflower mosaic virus (CaMV) (Hohn et al., (1982) Molecular Biology of Plant Tumors, (Academic Press, New York) pp. 549-560; U.S. Pat. No. 4,407,956), high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., (1987) Nature 327, 70-73), use of pollen as vector (WO 85/01856), or use of Agrobacterium tumefaciens or A. rhizogenes carrying a T-DNA plasmid in which DNA fragments are cloned. The T-DNA plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and a portion is stably integrated into the plant genome (Horsch et al. (1984) Science 233:496-498; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80, 4803).

The cell can include a nucleic acid of the invention which encodes a polypeptide, wherein the cells expresses a polypeptide of the invention. The cell can also include vector sequences, or the like. Furthermore, cells and transgenic plants which include any polypeptide or nucleic acid above or throughout this specification, e.g., produced by transduction of a vector of the invention, are an additional feature of the invention.

For long-term, high-yield production of recombinant proteins, stable expression can be used. Host cells transformed with a nucleotide sequence encoding a polypeptide of the invention are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein or fragment thereof produced by a recombinant cell may be secreted, membrane-bound, or contained intracellularly, depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides encoding mature proteins of the invention can be designed with signal sequences which direct secretion of the mature polypeptides through a prokaryotic or eukaryotic cell membrane.

Modified Amino Acids

Polypeptides of the invention may contain one or more modified amino acids. The presence of modified amino acids may be advantageous in, for example, increasing polypeptide half-life, reducing polypeptide antigenicity or toxicity, increasing polypeptide storage stability, or the like. Amino acid(s) are modified, for example, co-translationally or post-translationally during recombinant production or modified by synthetic or chemical means.

Non-limiting examples of a modified amino acid include incorporation or other use of acetylated amino acids, glycosylated amino acids, sulfated amino acids, prenylated (e.g., farnesylated, geranylgeranylated) amino acids, PEG modified (e.g., “PEGylated”) amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, etc. References adequate to guide one of skill in the modification of amino acids are replete throughout the literature.

Identification of Additional Factors

A transcription factor provided by the present invention can also be used to identify additional endogenous or exogenous molecules that can affect a phenotype or trait of interest. On the one hand, such molecules include organic (small or large molecules) and/or inorganic compounds that affect expression of (i.e., regulate) a particular transcription factor. Alternatively, such molecules include endogenous molecules that are acted upon either at a transcriptional level by a transcription factor of the invention to modify a phenotype as desired. For example, the transcription factors can be employed to identify one or more downstream gene with which is subject to a regulatory effect of the transcription factor. In one approach, a transcription factor or transcription factor homologue of the invention is expressed in a host cell, e.g, a transgenic plant cell, tissue or explant, and expression products, either RNA or protein, of likely or random targets are monitored, e.g., by hybridization to a microarray of nucleic acid probes corresponding to genes expressed in a tissue or cell type of interest, by two-dimensional gel electrophoresis of protein products, or by any other method known in the art for assessing expression of gene products at the level of RNA or protein. Alternatively, a transcription factor of the invention can be used to identify promoter sequences (i.e., binding sites) involved in the regulation of a downstream target. After identifying a promoter sequence, interactions between the transcription factor and the promoter sequence can be modified by changing specific nucleotides in the promoter sequence or specific amino acids in the transcription factor that interact with the promoter sequence to alter a plant trait. Typically, transcription factor DNA binding sites are identified by gel shift assays. After identifying the promoter regions, the promoter region sequences can be employed in double-stranded DNA arrays to identify molecules that affect the interactions of the transcription factors with their promoters (Bulyk et al. (1999) Nature Biotechnol. 17:573-577).

The identified transcription factors are also useful to identify proteins that modify the activity of the transcription factor. Such modification can occur by covalent modification, such as by phosphorylation, or by protein-protein (homo or -heteropolymer) interactions. Any method suitable for detecting protein-protein interactions can be employed. Among the methods that can be employed are co-immunoprecipitation, cross-linking and co-purification through gradients or chromatographic columns, and the two-hybrid yeast system.

The two-hybrid system detects protein interactions in vivo and is described in Chien, et al., (1991), Proc. Natl. Acad. Sci. USA 88, 9578-9582 and is commercially available from Clontech (Palo Alto, Calif.). In such a system, plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the TF polypeptide and the other consists of the transcription activator protein's activation domain fused to an unknown protein that is encoded by a cDNA that has been recombined into the plasmid as part of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., lacZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product. Then, the library plasmids responsible for reporter gene expression are isolated and sequenced to identify the proteins encoded by the library plasmids. After identifying proteins that interact with the transcription factors, assays for compounds that interfere with the TF protein-protein interactions can be preformed.

Identification of Modulators

In addition to the intracellular molecules described above, extracellular molecules that alter activity or expression of a transcription factor, either directly or indirectly, can be identified. For example, the methods can entail first placing a candidate molecule in contact with a plant or plant cell. The molecule can be introduced by topical administration, such as spraying or soaking of a plant, and then the molecule's effect on the expression or activity of the TF polypeptide or the expression of the polynucleotide monitored. Changes in the expression of the TF polypeptide can be monitored by use of polyclonal or monoclonal antibodies, gel electrophoresis or the like. Changes in the expression of the corresponding polynucleotide sequence can be detected by use of microarrays, Northern blots, quantitative PCR, or any other technique for monitoring changes in mRNA expression. These techniques are exemplified in Ausubel et al. (eds) Current Protocols in Molecular Biology, John Wiley & Sons (1998). Such changes in the expression levels can be correlated with modified plant traits and thus identified molecules can be useful for soaking or spraying on fruit, vegetable and grain crops to modify traits in plants.

Essentially any available composition can be tested for modulatory activity of expression or activity of any nucleic acid or polypeptide herein. Thus, available libraries of compounds such as chemicals, polypeptides, nucleic acids and the like can be tested for modulatory activity. Often, potential modulator compounds can be dissolved in aqueous or organic (e.g., DMSO-based) solutions for easy delivery to the cell or plant of interest in which the activity of the modulator is to be tested. Optionally, the assays are designed to screen large modulator composition libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays).

In one embodiment, high throughput screening methods involve providing a combinatorial library containing a large number of potential compounds (potential modulator compounds). Such “combinatorial chemical libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as target compounds.

A combinatorial chemical library can be, e.g., a collection of diverse chemical compounds generated by chemical synthesis or biological synthesis. For example, a combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (e.g., in one example, amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound of a set length). Exemplary libraries include peptide libraries, nucleic acid libraries, antibody libraries (see, e.g., Vaughn et al. (1996) Nature Biotechnol. 14:309-314 and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. Science (1996) 274:1520-1522 and U.S. Pat. No. 5,593,853), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), and small organic molecule libraries (see, e.g., benzodiazepines, Baum (1993) Chem Eng. News January 18, page 33; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337) and the like.

Preparation and screening of combinatorial or other libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res. 37:487-493 and Houghton et al. Nature (1991) 354:84-88). Other chemistries for generating chemical diversity libraries can also be used.

In addition, as noted, compound screening equipment for high-throughput screening is generally available, e.g., using any of a number of well known robotic systems that have also been developed for solution phase chemistries useful in assay systems. These systems include automated workstations including an automated synthesis apparatus and robotic systems utilizing robotic arms. Any of the above devices are suitable for use with the present invention, e.g., for high-throughput screening of potential modulators. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art.

Indeed, entire high throughput screening systems are commercially available. These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. Similarly, microfluidic implementations of screening are also commercially available.

The manufacturers of such systems provide detailed protocols the various high throughput. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like. The integrated systems herein, in addition to providing for sequence alignment and, optionally, synthesis of relevant nucleic acids, can include such screening apparatus to identify modulators that have an effect on one or more polynucleotides or polypeptides according to the present invention.

In some assays it is desirable to have positive controls to ensure that the components of the assays are working properly. At least two types of positive controls are appropriate. That is, known transcriptional activators or inhibitors can be incubated with cells/plants/etc. in one sample of the assay, and the resulting increase/decrease in transcription can be detected by measuring the resulting increase in RNA/protein expression, etc., according to the methods herein. It will be appreciated that modulators can also be combined with transcriptional activators or inhibitors to find modulators which inhibit transcriptional activation or transcriptional repression. Either expression of the nucleic acids and proteins herein or any additional nucleic acids or proteins activated by the nucleic acids or proteins herein, or both, can be monitored.

In an embodiment, the invention provides a method for identifying compositions that modulate the activity or expression of a polynucleotide or polypeptide of the invention. For example, a test compound, whether a small or large molecule, is placed in contact with a cell, plant (or plant tissue or explant), or composition comprising the polynucleotide or polypeptide of interest and a resulting effect on the cell, plant, (or tissue or explant) or composition is evaluated by monitoring, either directly or indirectly, one or more of: expression level of the polynucleotide or polypeptide, activity (or modulation of the activity) of the polynucleotide or polypeptide. In some cases, an alteration in a plant phenotype can be detected following contact of a plant (or plant cell, or tissue or explant) with the putative modulator, e.g., by modulation of expression or activity of a polynucleotide or polypeptide of the invention.

Subsequences

Also contemplated are uses of polynucleotides, also referred to herein as oligonucleotides, typically having at least 12 bases, preferably at least 15, more preferably at least 20, 30, or 50 bases, which hybridize under at least highly stringent (or ultra-high stringent or ultra-ultra-high stringent conditions) conditions to a polynucleotide sequence described above. The polynucleotides may be used as probes, primers, sense and antisense agents, and the like, according to methods as noted supra.

Subsequences of the polynucleotides of the invention, including polynucleotide fragments and oligonucleotides are useful as nucleic acid probes and primers. An oligonucleotide suitable for use as a probe or primer is at least about 15 nucleotides in length, more often at least about 18 nucleotides, often at least about 21 nucleotides, frequently at least about 30 nucleotides, or about 40 nucleotides, or more in length. A nucleic acid probe is useful in hybridization protocols, e.g., to identify additional polypeptide homologues of the invention, including protocols for microarray experiments. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods. See Sambrook and Ausubel, supra.

In addition, the invention includes an isolated or recombinant polypeptide including a subsequence of at least about 15 contiguous amino acids encoded by the recombinant or isolated polynucleotides of the invention. For example, such polypeptides, or domains or fragments thereof, can be used as immunogens, e.g., to produce antibodies specific for the polypeptide sequence, or as probes for detecting a sequence of interest. A subsequence can range in size from about 15 amino acids in length up to and including the full length of the polypeptide.

Introduction to the GARP Family

The acronym GARP was adopted to describe this family of transcription factors based on three of the founding members of the family: maize GOLDEN2, the ARR B class of Arabidopsis response regulator homologs, and Psr1 of Chlamydomonas reinhardtii (Riechmann et al. (2000) Science 290:2105-2110). These proteins share a putative DNA binding domain with limited homology to the myb superfamily of transcription factors (Sakai et al. (1998) Plant Cell Physiol. 39: 1232-1239), particularly to a family of Myb-related proteins that includes the circadian regulatory protein CCA1 (Riechmann et al. 2000) supra). Distant homology of this domain is also evident to the TEA DNA binding domain found in a number of regulatory genes from fungi, insects, and mammals (Burglin (1991) Cell 66: 11-12; Hall et al. (1998) Plant Cell 10: 925-36). The TEA domain is predicted to form two α helices that are implicated in DNA binding (Burglin (1991) supra). For simplicity, this putative DNA binding domain is referred to as the GARP domain. The GARP domain is a highly conserved stretch of 49-50 amino acids that begins with an invariant tryptophan residue and ends in a motif with the consensus sequence SHLQKYRL (SEQ ID NO: 2008), in which the first four amino acids appear to be invariant.

The founding member of the GARP family is the maize GOLDEN2 (G2) gene (Hall et al. (1998) supra). Maize uses the C4 pathway of photosynthesis, where photosynthetic carbon fixation reactions are segregated between the mesophyll and bundle sheath cells. The g2 mutation perturbs the specialized development of the bundle sheath cells and expression of the C4 photosynthetic pathway enzymes. Evidence provided by G2-GUS fusions indicates that G2 is localized to the nucleus. The GARP domain was subsequently found in a Chlamydomonas reinhardtii protein, PSR1, that is a nuclear-localized regulator of phosphorus metabolism (Wykoff et al. (1999) Proc. Natl. Acad. Sci. USA 96: 15336-15341) as well as in a tobacco protein submitted to GenBank as a transfactor, WREBP-1 (acc. no. AB017693).

Fifty-six GARP genes are present in Arabidopsis, and these fall into two major classes. The first class consists of proteins that contain the GARP domain as the only recognizable motif (44 genes). G2, PSR1, and WREBP-1 are of this type. The second class also contains an N terminal domain with similarity to bacterial response regulators (12 genes). These proteins have been termed ARR for Arabidopsis Response Regulator (Sakai et al. (1998) supra), or ARP for Arabidopsis Receiver-like Protein (acc. no. AJ005194).

The response regulator class of GARP proteins is of particular interest because of the growing evidence that phosphorelay signal transduction systems, with homology to prokaryotic two-component systems, are functional in plants. The simplest bacterial two-component systems consist of a sensor kinase and a response regulator protein. The sensor kinase autophosphorylates on a histidine residue, and the rate of autophosphorylation is modified by input from a sensor domain. The phosphate group is then transferred to an aspartate residue on the response regulator. In prokaryotes the response regulator is usually a transcription factor that activates downstream responses, although some response regulators have different modes of action. Phosphorelay systems of greater complexity are known, where the phosphate is passed through one or more intermediary phosphotransmitter proteins before phosphorylation of the response regulator. Other variations include proteins with fused sensor kinase and receiver domains (hybrid kinases), and the Arabidopsis ETR1 protein is a eukaryotic example of this class (for reviews see D'Agostino and Kieber (1999) Trends Biochem Sci. 24: 452-456); Chang and Stewart (1998) Plant Physiol. 117: 723-731). The response regulator class of GARP proteins is a subset of a group of putative Arabidopsis response regulators that has been termed the type-B response regulators. The type-A response regulators in contrast lack a putative DNA binding domain (D'Agostino and Kieber (1999) supra). The type-B response regulators are likely to be the functional equivalents of bacterial response regulators, which receive a signal from a sensor kinase and activate transcription. ARR type-B proteins have been shown to bind DNA (Sakai et al. (2000) Plant J. 24: 703-711; Lohrmann et al. (2001) Mol. Genet. Genomics 265: 2-13), and to interact with histidine phosphotransmitter proteins (Imamura et al. (1999) Plant Cell Physiol. 40: 733-742).

Recent work implicates the response regulator GARP (ARR type-B) proteins in cytokinin signal transduction. ARR1, ARR2, and ARR10 activate transcription of the cytokinin-regulated type-A ARR gene ARR6 in protoplasts (Hwang and Sheen (2001) Nature 413: 383-389). The cytokinin receptor CRE1 was recently found to be a histidine kinase with fused receiver domains (Inoue et al. (2001) Nature 409: 1060-1063). A signal transduction pathway is postulated where CRE1 initiates a phosphorelay, the signal is transduced to the nucleus through histidine phosphotransmitter proteins, and these proteins interact with ARR type-B proteins to release these proteins from putative repressors, allowing them to activate transcription. Among the genes induced are those encoding ARR type-A proteins, which are thought to serve as negative feedback regulators of the pathway (Hwang and Sheen (2001) supra).

It should be noted that one Arabidopsis protein with a GARP domain, AT1, was identified in a screen for clones affecting cell shape when overexpressed in Schizosaccharomyces pombe. Overexpression of AT1 caused disordered actin staining and cell elongation similar to the effects of overexpressing cytoskeletal components. On the basis of these results, AT1 was characterized as a putative cytoskeletal protein, and annotated as such in GenBank (Xia et al, (1996) Plant J. 10:761-769). However, the effects that AT1 overexpression produced could also be due to inappropriate activation of “S. pombe” genes. Because AT1 was the only annotated protein with a GARP domain in the database for some time, a number of Arabidopsis proteins with GARP domains were annotated as putative cytoskeletal proteins, even though the annotation of AT1 is tenuous.

G1435, SEQ ID NO: 1796, encoded by SEQ ID NO: 99, is an example of a GARP family transcription factor polypeptide. A number of sequences have been found in other plant species that are closely-related to G1435. Table 4 shows a number of polypeptides of the invention and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the Gene Identifier (“GID”; Column 2), the percent identity of the polypeptide in Column 1 to the full length G1435 polypeptide, SEQ ID NO: 1, as determined by a BLASTp analysis with a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915; Column 3), the amino acid residue coordinates for the respective conserved GARP domains, in amino acid coordinates beginning at the n-terminus, of each of the sequences (Column 4), the conserved GARP domain sequences of the respective polypeptides (Column 5); the SEQ ID NO: of each of the GARP domains (Column 6), and the percentage identity of the conserved GARP domain in Column 5 to the conserved GARP domain, SEQ ID NO: 1995, in the Arabidopsis G1435 sequence, SEQ ID NO: 1796 (Column 7; determined by BLASTp analysis as indicated above).

TABLE 4 Conserved domains and potentially valuable morphological traits of G1435 and closely related sequences Column 3 Percent identity of poly- peptide in Column 7 Column 1 Percent to G1435 identity Column 1 (identical of conserved Poly- residues/ Column 4 Column 6 domain in peptide total GARP SEQ ID Column 5 SEQ Column 2 number of domain in Column 5 NO: of to conserved ID Species/ residues amino acid Conserved GARP domain of NO: GID No. compared) coordinates GARP domain domain G1435 1796 At/G1435 100% 146-194 WTPQLHKRFVDVVAH 1995 100% (298/298) LGIKNAVPKTIMQLM (49/49) NVEGLTRENVASHLQ KYRL 1984 At/G2741 61% 149-197 WTPQLHKRFVDVVAH 1996 100% (205/331) LGIKNAVPKTIMQLM (49/49) NVEGLTRENVASHLQ KYRL 1986 Gm/G4243 53% 148-196 WTPQLHKRFVDVVAH 1997 100% (147/274) LGIKNAVPKTIMQLM (49/49) NVEGLTRENVASHLQ KYRL 1988 Gm/G4244 56% 149-197 WTPQLHKRFVDVVAH 1998 100% (141/250) LGIKNAVPKTIMQLM (49/49) NVEGLTRENVASHLQ KYRL 1994 Zm/G4240 50% 141-189 WTPQLHKRFVDVVAH 2001 97% (130/257) LGMKNAVPKTIMQLM (47/49) NVEGLTRENVASHLQ KYRL 1992 Os/G4241 56% 123-171 WTPQLHKRFVEVVAH 2000 95% (119/212) LGMKNAVPKTIMQLM (47/49) NVEGLTRENVASHLQ KYRL 1990 Le/G4245 46% 155-203 WTPQLHKRFIEVVAH 1999 91% (146/317) LGIKGAVPKTIMQLM (45/49) NVEGLTRENVAGHLQ KYRL Species abbreviations for Table 4: At - Arabidopsis thaliana; Gm - Glycine max; Le - Lycopersicon esculentum; Os - Oryza sativa; Zm - Zea mays.

Production of Transgenic Plants

Modification of Traits

The polynucleotides of the invention are favorably employed to produce transgenic plants with various traits, or characteristics, that have been modified in a desirable manner, e.g., to improve the seed characteristics of a plant. For example, alteration of expression levels or patterns (e.g., spatial or temporal expression patterns) of one or more of the transcription factors (or transcription factor homologues) of the invention, as compared with the levels of the same protein found in a control or a wild type plant, can be used to modify a plant's traits. An illustrative example of trait modification, improved characteristics, by altering expression levels of a particular transcription factor is described further in the Examples and the Sequence Listing.

Antisense and Cosuppression Approaches

In addition to expression of the nucleic acids of the invention as gene replacement or plant phenotype modification nucleic acids, the nucleic acids are also useful for sense and anti-sense suppression of expression, e.g., to down-regulate expression of a nucleic acid of the invention, e.g., as a further mechanism for modulating plant phenotype. That is, the nucleic acids of the invention, or subsequences or anti-sense sequences thereof, can be used to block expression of naturally occurring homologous nucleic acids. A variety of sense and anti-sense technologies are known in the art, e.g., as set forth in Lichtenstein and Nellen (1997) Antisense Technology: A Practical Approach IRL Press at Oxford University, Oxford, England. In general, sense or anti-sense sequences are introduced into a cell, where they are optionally amplified, e.g., by transcription. Such sequences include both simple oligonucleotide sequences and catalytic sequences such as ribozymes.

For example, a reduction or elimination of expression (i.e., a “knock-out”) of a transcription factor or transcription factor homologue polypeptide in a transgenic plant, e.g., to modify a plant trait, can be obtained by introducing an antisense construct corresponding to the polypeptide of interest as a cDNA. For antisense suppression, the transcription factor or homologue cDNA is arranged in reverse orientation (with respect to the coding sequence) relative to the promoter sequence in the expression vector. The introduced sequence need not be the full length cDNA or gene, and need not be identical to the cDNA or gene found in the plant type to be transformed. Typically, the antisense sequence need only be capable of hybridizing to the target gene or RNA of interest. Thus, where the introduced sequence is of shorter length, a higher degree of homology to the endogenous transcription factor sequence will be needed for effective antisense suppression. While antisense sequences of various lengths can be utilized, preferably, the introduced antisense sequence in the vector will be at least 30 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases. Preferably, the length of the antisense sequence in the vector will be greater than 100 nucleotides. Transcription of an antisense construct as described results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from the endogenous transcription factor gene in the plant cell.

Suppression of endogenous transcription factor gene expression can also be achieved using a ribozyme. Ribozymes are RNA molecules that possess highly specific endoribonuclease activity. The production and use of ribozymes are disclosed in U.S. Pat. No. 4,987,071 and U.S. Pat. No. 5,543,508. Synthetic ribozyme sequences including antisense RNAs can be used to confer RNA cleaving activity on the antisense RNA, such that endogenous mRNA molecules that hybridize to the antisense RNA are cleaved, which in turn leads to an enhanced antisense inhibition of endogenous gene expression.

Vectors in which RNA encoded by a transcription factor or transcription factor homologue cDNA is over-expressed can also be used to obtain co-suppression of a corresponding endogenous gene, e.g., in the manner described in U.S. Pat. No. 5,231,020 to Jorgensen. Such co-suppression (also termed sense suppression) does not require that the entire transcription factor cDNA be introduced into the plant cells, nor does it require that the introduced sequence be exactly identical to the endogenous transcription factor gene of interest. However, as with antisense suppression, the suppressive efficiency will be enhanced as specificity of hybridization is increased, e.g., as the introduced sequence is lengthened, and/or as the sequence similarity between the introduced sequence and the endogenous transcription factor gene is increased.

Vectors expressing an untranslatable form of the transcription factor mRNA, e.g., sequences comprising one or more stop codon, or nonsense mutation) can also be used to suppress expression of an endogenous transcription factor, thereby reducing or eliminating it's activity and modifying one or more traits. Methods for producing such constructs are described in U.S. Pat. No. 5,583,021. Preferably, such constructs are made by introducing a premature stop codon into the transcription factor gene. Alternatively, a plant trait can be modified by gene silencing using double-strand RNA (Sharp (1999) Genes Dev. 13: 139-141).

Another method for abolishing the expression of a gene is by insertion mutagenesis using the T-DNA of Agrobacterium tumefaciens. After generating the insertion mutants, the mutants can be screened to identify those containing the insertion in a transcription factor or transcription factor homologue gene. Plants containing a single transgene insertion event at the desired gene can be crossed to generate homozygous plants for the mutation (Koncz et al. (1992) Methods in Arabidopsis Research, World Scientific).

Alternatively, a plant phenotype can be altered by eliminating an endogenous gene, such as a transcription factor or transcription factor homologue, e.g., by homologous recombination (Kempin et al. (1997) Nature 389:802).

A plant trait can also be modified by using the cre-lox system (for example, as described in U.S. Pat. No. 5,658,772). A plant genome can be modified to include first and second lox sites that are then contacted with a Cre recombinase. If the lox sites are in the same orientation, the intervening DNA sequence between the two sites is excised. If the lox sites are in the opposite orientation, the intervening sequence is inverted.

The polynucleotides and polypeptides of this invention can also be expressed in a plant in the absence of an expression cassette by manipulating the activity or expression level of the endogenous gene by other means. For example, by ectopically expressing a gene by T-DNA activation tagging (Ichikawa et al. (1997) Nature 390 698-701; Kakimoto et al. (1996) Science 274: 982-985). This method entails transforming a plant with a gene tag containing multiple transcriptional enhancers and once the tag has inserted into the genome, expression of a flanking gene coding sequence becomes deregulated. In another example, the transcriptional machinery in a plant can be modified so as to increase transcription levels of a polynucleotide of the invention (See, e.g., PCT Publications WO 96/06166 and WO 98/53057 which describe the modification of the DNA binding specificity of zinc finger proteins by changing particular amino acids in the DNA binding motif).

The transgenic plant can also include the machinery necessary for expressing or altering the activity of a polypeptide encoded by an endogenous gene, for example by altering the phosphorylation state of the polypeptide to maintain it in an activated state.

Transgenic plants (or plant cells, or plant explants, or plant tissues) incorporating the polynucleotides of the invention and/or expressing the polypeptides of the invention can be produced by a variety of well established techniques as described above. Following construction of a vector, most typically an expression cassette, including a polynucleotide, e.g., encoding a transcription factor or transcription factor homologue, of the invention, standard techniques can be used to introduce the polynucleotide into a plant, a plant cell, a plant explant or a plant tissue of interest. Optionally, the plant cell, explant or tissue can be regenerated to produce a transgenic plant.

The plant can be any higher plant, including gymnosperms, monocotyledonous and dicotyledonous plants. Suitable protocols are available for Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage, radish, rapeseed, broccoli, etc.), Curcurbitaceae (melons and cucumber), Gramineae (wheat, corn, rice, barley, millet, etc.), Solanaceae (potato, tomato, tobacco, peppers, etc.), and various other crops. See protocols described in Ammirato et al. (1984) Handbook of Plant Cell Culture—Crop Species. Macmillan Publ. Co. Shimamoto et al. (1989) Nature 338:274-276; Fromm et al. (1990) Bio/Technology 8:833-839; and Vasil et al. (1990) Bio/Technology 8:429-434.

Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells is now routine, and the selection of the most appropriate transformation technique will be determined by the practitioner. The choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods can include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium tumeficiens mediated transformation. Transformation means introducing a nucleotide sequence in a plant in a manner to cause stable or transient expression of the sequence.

Successful examples of the modification of plant characteristics by transformation with cloned sequences which serve to illustrate the current knowledge in this field of technology, and which are herein incorporated by reference, include: U.S. Pat. Nos. 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871; 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369 and 5,610,042.

Following transformation, plants are preferably selected using a dominant selectable marker incorporated into the transformation vector. Typically, such a marker will confer antibiotic or herbicide resistance on the transformed plants, and selection of transformants can be accomplished by exposing the plants to appropriate concentrations of the antibiotic or herbicide.

After transformed plants are selected and grown to maturity, those plants showing a modified trait are identified. The modified trait can be any of those traits described above. Additionally, to confirm that the modified trait is due to changes in expression levels or activity of the polypeptide or polynucleotide of the invention can be determined by analyzing mRNA expression using Northern blots, RT-PCR or microarrays, or protein expression using immunoblots or Western blots or gel shift assays.

Integrated Systems—Sequence Identity

Additionally, the present invention may be an integrated system, computer or computer readable medium that comprises an instruction set for determining the identity of one or more sequences in a database. In addition, the instruction set can be used to generate or identify sequences that meet any specified criteria. Furthermore, the instruction set may be used to associate or link certain functional benefits, such improved characteristics, with one or more identified sequence.

For example, the instruction set can include, e.g., a sequence comparison or other alignment program, e.g., an available program such as, for example, the Wisconsin Package Version 10.0, such as BLAST, FASTA, PILEUP, FINDPATTERNS or the like (GCG, Madison, Wis.). Public sequence databases such as GenBank, EMBL, Swiss-Prot and PIR or private sequence databases such as PhytoSeq (Incyte Pharmaceuticals, Palo Alto, Calif.) can be searched.

Alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85: 2444, by computerized implementations of these algorithms. After alignment, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window can be a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 contiguous positions. A description of the method is provided in Ausubel et al., supra.

A variety of methods of determining sequence relationships can be used, including manual alignment and computer assisted sequence alignment and analysis. This later approach is a preferred approach in the present invention, due to the increased throughput afforded by computer assisted methods. As noted above, a variety of computer programs for performing sequence alignment are available, or can be produced by one of skill.

One example algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) supra).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence (and, therefore, in this context, homologous) if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, or less than about 0.01, and or even less than about 0.001. An additional example of a useful sequence alignment algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. The program can align, e.g., up to 300 sequences of a maximum length of 5,000 letters.

The integrated system, or computer typically includes a user input interface allowing a user to selectively view one or more sequence records corresponding to the one or more character strings, as well as an instruction set which aligns the one or more character strings with each other or with an additional character string to identify one or more region of sequence similarity. The system may include a link of one or more character strings with a particular phenotype or gene function. Typically, the system includes a user readable output element which displays an alignment produced by the alignment instruction set.

The methods of this invention can be implemented in a localized or distributed computing environment. In a distributed environment, the methods may implemented on a single computer comprising multiple processors or on a multiplicity of computers. The computers can be linked, e.g. through a common bus, but more preferably the computer(s) are nodes on a network. The network can be a generalized or a dedicated local or wide-area network and, in certain preferred embodiments, the computers may be components of an intra-net or an internet.

Thus, the invention provides methods for identifying a sequence similar or homologous to one or more polynucleotides as noted herein, or one or more target polypeptides encoded by the polynucleotides, or otherwise noted herein and may include linking or associating a given plant phenotype or gene function with a sequence. In the methods, a sequence database is provided (locally or across an inter or intra net) and a query is made against the sequence database using the relevant sequences herein and associated plant phenotypes or gene functions.

Any sequence herein can be entered into the database, before or after querying the database. This provides for both expansion of the database and, if done before the querying step, for insertion of control sequences into the database. The control sequences can be detected by the query to ensure the general integrity of both the database and the query. As noted, the query can be performed using a web browser based interface. For example, the database can be a centralized public database such as those noted herein, and the querying can be done from a remote terminal or computer across an internet or intranet.

EXAMPLES

The following examples are intended to illustrate but not limit the present invention.

Example I Full Length Gene Identification and Cloning

Putative transcription factor sequences (genomic or ESTs) related to known transcription factors were identified in the Arabidopsis thaliana GenBank database using the tblastn sequence analysis program using default parameters and a P-value cutoff threshold of −4 or −5 or lower, depending on the length of the query sequence. Putative transcription factor sequence hits were then screened to identify those containing particular sequence strings. If the sequence hits contained such sequence strings, the sequences were confirmed as transcription factors.

Alternatively, Arabidopsis thaliana cDNA libraries derived from different tissues or treatments, or genomic libraries were screened to identify novel members of a transcription family using a low stringency hybridization approach. Probes were synthesized using gene specific primers in a standard PCR reaction (annealing temperature 60° C.) and labeled with ³²P dCTP using the High Prime DNA Labeling Kit (Boehringer Mannheim). Purified radiolabelled probes were added to filters immersed in Church hybridization medium (0.5 M NaPO₄ pH 7.0, 7% SDS, 1% w/v bovine serum albumin) and hybridized overnight at 60° C. with shaking. Filters were washed two times for 45 to 60 minutes with 1×SCC, 1% SDS at 60° C.

To identify additional sequence 5′ or 3′ of a partial cDNA sequence in a cDNA library, 5′ and 3′ rapid amplification of cDNA ends (RACE) was performed using the Marathon™ cDNA amplification kit (Clontech, Palo Alto, Calif.). Generally, the method entailed first isolating poly(A) mRNA, performing first and second strand cDNA synthesis to generate double stranded cDNA, blunting cDNA ends, followed by ligation of the Marathon™ Adaptor to the cDNA to form a library of adaptor-ligated ds cDNA.

Gene-specific primers were designed to be used along with adaptor specific primers for both 5′ and 3′ RACE reactions. Nested primers, rather than single primers, were used to increase PCR specificity. Using 5′ and 3′ RACE reactions, 5′ and 3′ RACE fragments were obtained, sequenced and cloned. The process can be repeated until 5′ and 3′ ends of the full-length gene were identified. Then the full-length cDNA was generated by PCR using primers specific to 5′ and 3′ ends of the gene by end-to-end PCR.

Example II Construction of Expression Vectors

The sequence was amplified from a genomic or cDNA library using primers specific to sequences upstream and downstream of the coding region. The expression vector was pMEN20 or pMEN65, which are both derived from pMON316 (Sanders et al, (1987) Nucleic Acids Res. 15:1543-58) and contain the CaMV 35S promoter to express transgenes. To clone the sequence into the vector, both pMEN20 and the amplified DNA fragment were digested separately with SalI and NotI restriction enzymes at 37° C. for 2 hours. The digestion products were subject to electrophoresis in a 0.8% agarose gel and visualized by ethidium bromide staining. The DNA fragments containing the sequence and the linearized plasmid were excised and purified by using a Qiaquick gel extraction kit (Qiagen, CA). The fragments of interest were ligated at a ratio of 3:1 (vector to insert). Ligation reactions using T4 DNA ligase (New England Biolabs, MA) were carried out at 16° C. for 16 hours. The ligated DNAs were transformed into competent cells of the E. coli strain DH5alpha by using the heat shock method. The transformations were plated on LB plates containing 50 mg/l kanamycin (Sigma).

Individual colonies were grown overnight in five milliliters of LB broth containing 50 mg/l kanamycin at 37° C. Plasmid DNA was purified by using Qiaquick Mini Prep kits (Qiagen, CA).

Example III Transformation of Agrobacterium with the Expression Vector

After the plasmid vector containing the gene was constructed, the vector was used to transform Agrobacterium tumefaciens cells expressing the gene products. The stock of Agrobacterium tumefaciens cells for transformation was made as described by Nagel et al. (1990) FEMS MicroBiol. Letts. 67: 325-328. Agrobacterium strain ABI was grown in 250 ml LB medium (Sigma) overnight at 28° C. with shaking until an absorbance (A₆₀₀) of 0.5-1.0 was reached. Cells were harvested by centrifugation at 4,000×g for 15 min at 4° C. Cells were then resuspended in 250 μl chilled buffer (1 mM HEPES, pH adjusted to 7.0 with KOH). Cells were centrifuged again as described above and resuspended in 125 μl chilled buffer. Cells were then centrifuged and resuspended two more times in the same HEPES buffer as described above at a volume of 100 μl and 750 μl, respectively. Resuspended cells were then distributed into 40 μl aliquots, quickly frozen in liquid nitrogen, and stored at −80° C.

Agrobacterium cells were transformed with plasmids prepared as described above following the protocol described by Nagel et al. For each DNA construct to be transformed, 50-100 ng DNA (generally resuspended in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was mixed with 40 μl of Agrobacterium cells. The DNA/cell mixture was then transferred to a chilled cuvette with a 2 mm electrode gap and subject to a 2.5 kV charge dissipated at 25 μF and 200° F. using a Gene Pulser II apparatus (Bio-Rad). After electroporation, cells were immediately resuspended in 1.0 ml LB and allowed to recover without antibiotic selection for 2-4 hours at 28° C. in a shaking incubator. After recovery, cells were plated onto selective medium of LB broth containing 100 μg/ml spectinomycin (Sigma) and incubated for 24-48 hours at 28° C. Single colonies were then picked and inoculated in fresh medium. The presence of the plasmid construct was verified by PCR amplification and sequence analysis.

Example IV Transformation of Arabidopsis Plants with Agrobacterium tumefaciens with Expression Vector

After transformation of Agrobacterium tumefaciens with plasmid vectors containing the gene, single Agrobacterium colonies were identified, propagated, and used to transform Arabidopsis plants. Briefly, 500 ml cultures of LB medium containing 50 mg/l kanamycin were inoculated with the colonies and grown at 28° C. with shaking for 2 days until an absorbance (A₆₀₀) of >2.0 is reached. Cells were then harvested by centrifugation at 4,000×g for 10 min, and resuspended in infiltration medium (½× Murashige and Skoog salts (Sigma), 1× Gamborg's B-5 vitamins (Sigma), 5.0% (w/v) sucrose (Sigma), 0.044 μM benzylamino purine (Sigma), 200 μl/L Silwet L-77 (Lehle Seeds) until an absorbance (A₆₀₀) of 0.8 was reached.

Prior to transformation, Arabidopsis thaliana seeds (ecotype Columbia) were sown at a density of ˜10 plants per 4″ pot onto Pro-Mix BX potting medium (Hummert International) covered with fiberglass mesh (18 mm×16 mm). Plants were grown under continuous illumination (50-75 μE/m²/sec) at 22-23° C. with 65-70% relative humidity. After about 4 weeks, primary inflorescence stems (bolts) are cut off to encourage growth of multiple secondary bolts. After flowering of the mature secondary bolts, plants were prepared for transformation by removal of all siliques and opened flowers.

The pots were then immersed upside down in the mixture of Agrobacterium infiltration medium as described above for 30 sec, and placed on their sides to allow draining into a 1′×2′ flat surface covered with plastic wrap. After 24 h, the plastic wrap was removed and pots are turned upright. The immersion procedure was repeated one week later, for a total of two immersions per pot. Seeds were then collected from each transformation pot and analyzed following the protocol described below.

Example V Identification of Arabidopsis Primary Transformants

Seeds collected from the transformation pots were sterilized essentially as follows. Seeds were dispersed into in a solution containing 0.1% (v/v) Triton X-100 (Sigma) and sterile H₂O and washed by shaking the suspension for 20 min. The wash solution was then drained and replaced with fresh wash solution to wash the seeds for 20 min with shaking. After removal of the second wash solution, a solution containing 0.1% (v/v) Triton X-100 and 70% ethanol (Equistar) was added to the seeds and the suspension was shaken for 5 min. After removal of the ethanol/detergent solution, a solution containing 0.1% (v/v) Triton X-100 and 30% (v/v) bleach (Clorox) was added to the seeds, and the suspension was shaken for 10 min. After removal of the bleach/detergent solution, seeds were then washed five times in sterile distilled H₂O. The seeds were stored in the last wash water at 4° C. for 2 days in the dark before being plated onto antibiotic selection medium (1× Murashige and Skoog salts (pH adjusted to 5.7 with 1M KOH), 1× Gamborg's B-5 vitamins, 0.9% phytagar (Life Technologies), and 50 mg/l kanamycin). Seeds were germinated under continuous illumination (50-75 μE/m²/sec) at 22-23° C. After 7-10 days of growth under these conditions, kanamycin resistant primary transformants (T₁ generation) were visible and obtained. These seedlings were transferred first to fresh selection plates where the seedlings continued to grow for 3-5 more days, and then to soil (Pro-Mix BX potting medium).

Primary transformants were crossed and progeny seeds (T₂) collected; kanamycin resistant seedlings were selected and analyzed. The expression levels of the recombinant polynucleotides in the transformants vary from about a 5% expression level increase to a least a 100% expression level increase. Similar observations are made with respect to polypeptide level expression.

Example VI Identification of Arabidopsis Plants with Transcription Factor Gene Knockouts

The screening of insertion mutagenized Arabidopsis collections for null mutants in a known target gene was essentially as described in Krysan et al (1999) Plant Cell 11:2283-2290. Briefly, gene-specific primers, nested by 5-250 base pairs to each other, were designed from the 5′ and 3′ regions of a known target gene. Similarly, nested sets of primers were also created specific to each of the T-DNA or transposon ends (the “right” and “left” borders). All possible combinations of gene specific and T-DNA/transposon primers were used to detect by PCR an insertion event within or close to the target gene. The amplified DNA fragments were then sequenced which allows the precise determination of the T-DNA/transposon insertion point relative to the target gene. Insertion events within the coding or intervening sequence of the genes were deconvoluted from a pool comprising a plurality of insertion events to a single unique mutant plant for functional characterization. The method is described in more detail in Yu and Adam, U.S. application Ser. No. 09/177,733 filed Oct. 23, 1998.

Example VII Identification of Modified Phenotype in Overexpressor or Gene Knockout Plants

Experiments were performed to identify those transformants or knockouts that exhibited modified biochemical characteristics. Among the biochemicals that were assayed were insoluble sugars, such as arabinose, fucose, galactose, mannose, rhamnose or xylose or the like; prenyl lipids, such as lutein, β-carotene, xanthophyll-1, xanthophyll-2, chlorophylls A or B, or α-, δ- or γ-tocopherol or the like; fatty acids, such as 16:0 (palmitic acid), 16:1 (palmitoleic acid), 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (linoleic acid), 20:0, 18:3 (linolenic acid), 20:1 (eicosenoic acid), 20:2, 22:1 (erucic acid) or the like; waxes, such as by altering the levels of C29, C31, or C33 alkanes; sterols, such as brassicasterol, campesterol, stigmasterol, sitosterol or stigmastanol or the like, glucosinolates, protein or oil levels

Fatty acids were measured using two methods depending on whether the tissue was from leaves or seeds. For leaves, lipids were extracted and esterified with hot methanolic H2SO4 and partitioned into hexane from methanolic brine. For seed fatty acids, seeds were pulverized and extracted in methanol:heptane:toluene:2,2-dimethoxypropane:H₂SO₄ (39:34:20:5:2) for 90 minutes at 80° C. After cooling to room temperature the upper phase, containing the seed fatty acid esters, was subjected to GC analysis. Fatty acid esters from both seed and leaf tissues were analyzed with a Supelco SP-2330 column.

Glucosinolates were purified from seeds or leaves by first heating the tissue at 95° C. for 10 minutes. Preheated ethanol:water (50:50) is and after heating at 95° C. for a further 10 minutes, the extraction solvent is applied to a DEAE Sephadex column which had been previously equilibrated with 0.5 M pyridine acetate. Desulfoglucosinolates were eluted with 300 μl water and analyzed by reverse phase HPLC monitoring at 226 nm.

For wax alkanes, samples were extracted using an identical method as fatty acids and extracts were analyzed on a HP 5890 GC coupled with a 5973 MSD. Samples were chromatographed on a J&W DB35 mass spectrometer (J&W Scientific).

To measure prenyl lipids levels, seeds or leaves were pulverized with 1 to 2% pyrogallol as an antioxidant. For seeds, extracted samples were filtered and a portion removed for tocopherol and carotenoid/chlorophyll analysis by HPLC. The remaining material was saponified for sterol determination. For leaves, an aliquot was removed and diluted with methanol and chlorophyll A, chlorophyll B, and total carotenoids measured by spectrophotometry by determining absorbance at 665.2 nm, 652.5 nm, and 470 nm. An aliquot was removed for tocopherol and carotenoid/chlorophyll composition by HPLC using a Waters μBondapak® C18 column (4.6 mm×150 mm). The remaining methanolic solution was saponified with 10% KOH at 80° C. for one hour. The samples were cooled and diluted with a mixture of methanol and water. A solution of 2% methylene chloride in hexane was mixed in and the samples were centrifuged. The aqueous methanol phase was again re-extracted 2% methylene chloride in hexane and, after centrifugation, the two upper phases were combined and evaporated. 2% methylene chloride in hexane was added to the tubes and the samples were then extracted with one ml of water. The upper phase was removed, dried, and resuspended in 400 μl of 2% methylene chloride in hexane and analyzed by gas chromatography using a 50 m DB-5 ms (0.25 mm ID, 0.25 um phase, J&W Scientific).

Insoluble sugar levels were measured by the method essentially described by Reiter et al. Plant J. 12:335-345. This method analyzes the neutral sugar composition of cell wall polymers found in Arabidopsis leaves. Soluble sugars were separated from sugar polymers by extracting leaves with hot 70% ethanol. The remaining residue containing the insoluble polysaccharides was then acid hydrolyzed with allose added as an internal standard. Sugar monomers generated by the hydrolysis were then reduced to the corresponding alditols by treatment with NaBH₄, then were acetylated to generate the volatile alditol acetates which were then analyzed by GC-FID. Identity of the peaks was determined by comparing the retention times of known sugars converted to the corresponding alditol acetates with the retention times of peaks from wild-type plant extracts. Alditol acetates were analyzed on a Supelco SP-2330 capillary column (30 m×250 um×0.2 um) using a temperature program beginning at 180° C. for 2 minutes followed by an increase to 220° C. in 4 minutes. After holding at 220° C. for 10 minutes, the oven temperature is increased to 240° C. in 2 minutes and held at this temperature for 10 minutes and brought back to room temperature.

To identify plants with alterations in total seed oil or protein content, 150 mg of seeds from T2 progeny plants were subjected to analysis by Near Infrared Reflectance (NIR) using a Foss NirSystems Model 6500 with a spinning cup transport system.

Experiments were performed to identify those transformants or knockouts that exhibited an improved pathogen tolerance. For such studies, the transformants were exposed to biotropic fungal pathogens, such as Erysiphe orontii, and necrotropic fungal pathogens, such as Fusarium oxysporum. Fusarium oxysporum isolates cause vascular wilts and damping off of various annual vegetables, perennials and weeds (Mauch-Mani and Slusarenko (1994) Mol. Plant-Microbe Interact. 7: 378-383). For Fusarium oxysporum experiments, plants grown on Petri dishes were sprayed with a fresh spore suspension of F. oxysporum. The spore suspension was prepared as follows: A plug of fungal hyphae from a plate culture was placed on a fresh potato dextrose agar plate and allowed to spread for one week. 5 ml sterile water was then added to the plate, swirled, and pipetted into 50 ml Armstrong Fusarium medium. Spores were grown overnight in Fusarium medium and then sprayed onto plants using a Preval paint sprayer. Plant tissue was harvested and frozen in liquid nitrogen 48 hours post infection.

Erysiphe orontii is a causal agent of powdery mildew. For Erysiphe orontii experiments, plants were grown approximately 4 weeks in a greenhouse under 12 hour light (20 C, ˜30% relative humidity (rh)). Individual leaves were infected with E. orontii spores from infected plants using a camel's hair brush, and the plants were transferred to a Percival growth chamber (20 C, 80% rh.). Plant tissue was harvested and frozen in liquid nitrogen 7 days post infection.

Botrytis cinerea is a necrotrophic pathogen. Botrytis cinerea was grown on potato dextrose agar in the light. A spore culture was made by spreading 10 ml of sterile water on the fungus plate, swirling and transferring spores to 10 ml of sterile water. The spore inoculum (approx. 105 spores/ml) was used to spray 10 day-old seedlings grown under sterile conditions on MS (-sucrose) media. Symptoms were evaluated every day up to approximately 1 week.

Infection with bacterial pathogens Pseudomonas syringae pv maculicola strain 4326 and pv maculicola strain 4326 was performed by hand inoculation at two doses. Administration of two inoculation doses allows the differentiation between plants with enhanced susceptibility and plants with enhanced resistance to the pathogen. Plants were grown for 3 weeks in the greenhouse, then transferred to the growth chamber for the remainder of their growth. Psm ES4326 was hand inoculated with 1 ml syringe on 3 fully-expanded leaves per plant (4½ wk old), using at least 9 plants per overexpressing line at two inoculation doses, OD=0.005 and OD=0.0005. Disease scoring occurred at day 3 post-inoculation with pictures of the plants and leaves taken in parallel

In some instances, expression patterns of the pathogen induced genes (such as defense genes) were monitored by microarray experiments. cDNAs were generated by PCR and resuspended at a final concentration of ˜100 ng/μl in 3×SSC or 150 mM Na-phosphate (Eisen and Brown (1999) Meth. in Enzymol. 303:179-205). The cDNAs were spotted on microscope glass slides coated with polylysine. The prepared cDNAs were aliquoted into 384 well plates and spotted on the slides using an x-y-z gantry (OmniGrid) purchased from GeneMachines (Menlo Park, Calif.) outfitted with quill type pins purchased from Telechem International (Sunnyvale, Calif.). After spotting, the arrays were cured for a minimum of one week at room temperature, rehydrated and blocked following the protocol recommended by Eisen and Brown (1999).

Sample total RNA (10 μg) samples were labeled using fluorescent Cy3 and Cy5 dyes. Labeled samples were resuspended in 4×SSC/0.03% SDS/4 μg salmon sperm DNA/2 μg tRNA/50 mM Na-pyrophosphate, heated for 95° C. for 2.5 minutes, spun down and placed on the array. The array was then covered with a glass coverslip and placed in a sealed chamber. The chamber was then kept in a water bath at 62° C. overnight. The arrays were washed as described in Eisen and Brown (1999) and scanned on a General Scanning ScanArray™ 3000 laser scanner. The resulting files are subsequently quantified using Imagene a software purchased from BioDiscovery (Los Angeles, Calif.).

Measurement of Photosynthesis.

Photosynthesis was measured using a LICOR LI-6400 (Li-Cor® Biosciences, Lincoln, Nebr.). The LI-6400 used infrared gas analyzers to measure carbon dioxide to generate a photosynthesis measurement. It was based upon the difference of the CO₂ reference (the amount put into the chamber) and the CO₂ sample (the amount that leaves the chamber). Since photosynthesis is the process of converting CO₂ to carbohydrates, we expected to see a decrease in the amount of CO₂ sample. From this difference, a photosynthesis rate could be generated. In some cases, respiration may occur and an increase in CO₂ detected. To perform measurements, the LI-6400 as set-up and calibrated as per LI-6400 standard directions. Photosynthesis was measured in the youngest, most fully expanded leaf at 300 and 1000 ppm CO₂ using a metal halide light source. This light source provided about 700 μE m⁻² s⁻¹.

Environmental Stress Tolerance.

Experiments were performed to identify those transformants or knockouts that exhibited an improved environmental stress tolerance. For such studies, the transformants were exposed to a variety of environmental stresses. Plants were exposed to chilling stress (6 hour exposure to 4°-8° C.), heat stress (6 hour exposure to 32°-37° C.), high salt stress (germination in 150 mM NaCl or a 6 hour exposure of plants to 200 mM NaCl), drought stress (withholding of water for 168 hours), hyperosmotic stress (for example, germination in 9.4% sucrose or a 6 hour exposure to 3 M mannitol), desiccation, or nutrient limitation (nitrogen, phosphate, and potassium) (Nitrogen: all components of MS medium remained constant except N was reduced to 20 mg/L of NH₄NO₃, or Phosphate: All components of MS medium except KH₂PO₄, which was replaced by K₂SO₄, Potassium: All components of MS medium except removal of KNO₃ and KH₂PO₄, which were replaced by NaH₄PO₄). For analysis of ability to tolerate desiccation (a plate-based water deprivation assay), seedlings were grown for 14 days on MS+Vitamins+1% sucrose at 22° C. Plates were opened in the sterile hood for 3 hr for hardening and then seedlings were removed from the media and let dry for two hours in the hood. After this time the plants were transferred back to plates and incubated at 22° C. for recovery. The plants were then evaluated after five days.

Soil-based drought assays. Seeds were sterilized by a 2 minute ethanol treatment followed by 20 minutes in 30% bleach/0.01% Tween and five washes in distilled water. Seeds were sown to MS agar in 0.1% agarose and stratified for three days at 4° C., before transfer to growth cabinets with a temperature of 22° C. After seven days of growth on selection plates, seedlings were transplanted to 3.5 inch diameter clay pots containing 80 g of a 50:50 mix of vermiculite:perlite topped with 80 g of ProMix. Typically, each pot contains 14 seedlings, and plants of the transgenic line being tested were in separate pots to the wild-type controls. Pots containing the transgenic line versus control pots were interspersed in the growth room, maintained under 24-hour light conditions (18-23° C., and 90-100 μE m⁻² s⁻¹) and watered for a period of 14 days. Water was then withheld and pots were placed on absorbent paper for a period of 8-10 days to apply a drought treatment. After this period, a visual qualitative “drought score” from 0-6 was assigned to record the extent of visible drought stress symptoms. A score of “6” corresponded to no visible symptoms whereas a score of “0” corresponded to extreme wilting and the leaves having a “crispy” texture. At the end of the drought period, pots were re-watered and scored after 5-6 days; the number of surviving plants in each pot was counted, and the proportion of the total plants in the pot that survived was calculated.

In a given experiment, we typically compared 6 or more pots of a transgenic line with 6 or more pots of the appropriate control. The mean drought score and mean proportion of plants surviving (survival rate) were calculated for both the transgenic line and the wild-type pots. In each case a p-value* was calculated, which indicated the significance of the difference between the two mean values. The results for each transgenic line across each planting for a particular project were then presented in a results table.

For the assays where control and experimental plants were in separate pots, survival was analyzed with a logistic regression to account for the fact that the random variable is a proportion between 0 and 1. The reported p-value was the significance of the experimental proportion was to the control, based upon regressing the logit-transformed data.

Drought score, being an ordered factor with no real numeric meaning, is analyzed with a non-parametric test between the experimental and control groups. The p-value was calculated with a Mann-Whitney rank-sum test.

Experiments were performed to identify those transformants or knockouts that exhibited a modified structure and development characteristics. For such studies, the transformants were observed by eye to identify novel structural or developmental characteristics associated with the ectopic expression of the polynucleotides or polypeptides of the invention.

Experiments were performed to identify those transformants or knockouts that exhibited modified sugar-sensing. For such studies, seeds from transformants were germinated on media containing 5% glucose or 9.4% sucrose which normally partially restrict hypocotyl elongation. Plants with altered sugar sensing may have either longer or shorter hypocotyls than normal plants when grown on this media. Additionally, other plant traits may be varied such as root mass.

Flowering time was measured by the number of rosette leaves present when a visible inflorescence of approximately 3 cm is apparent Rosette and total leaf number on the progeny stem are tightly correlated with the timing of flowering (Koornneef et al (1991) Mol. Gen. Genet 229:57-66. The vernalization response was measured. For vernalization treatments, seeds were sown to MS agar plates, sealed with micropore tape, and placed in a 4° C. cold room with low light levels for 6-8 weeks. The plates were then transferred to the growth rooms alongside plates containing freshly sown non-vernalized controls. Rosette leaves were counted when a visible inflorescence of approximately 3 cm was apparent.

Table 5 shows exemplary modified phenotypes observed for particular overexpressor or knockout plants. Modified phenotypes observed for particular overexpressor or knockout plants were provided in Appendix A in U.S. priority application Ser. No. 09/713,994, filed Nov. 16, 2000 (Appendix A is herein incorporated by reference in its entirety). For a particular overexpressor that shows a less beneficial characteristic, it may be more useful to select a plant with a decreased expression of the particular transcription factor. For a particular knockout that shows a less beneficial characteristic, it may be more useful to select a plant with an increased expression of the particular transcription factor.

Example VIII Identification of Homologous Sequences

Homologous sequences from Arabidopsis and plant species other than Arabidopsis were identified using database sequence search tools, such as the Basic Local Alignment Search Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucl. Acid Res. 25: 3389-3402). The tblastx sequence analysis programs were employed using the BLOSUM-62 scoring matrix (Henikoff, S. and Henikoff, J. G. (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919).

Identified Arabidopsis homologous sequences are provided in Tables 4 and 5 and included in the Sequence Listing. The percent sequence identity among these sequences is as low as 46% sequence identity. Additionally, the entire NCBI GenBank database was filtered for sequences from all plants except Arabidopsis thaliana by selecting all entries in the NCBI GenBank database associated with NCBI taxonomic ID 33090 (Viridiplantae; all plants) and excluding entries associated with taxonomic ID 3701 (Arabidopsis thaliana). These sequences were compared to sequences representing genes of SEQ IDs Nos. 1-54 on Sep. 26, 2000 using the Washington University TBLASTX algorithm (version 2.0a19 MP). For each gene of SEQ IDs Nos. 1-54, individual comparisons were ordered by probability score (P-value), where the score reflects the probability that a particular alignment occurred by chance. For example, a score of 3.6e-40 is 3.6×10⁻⁴⁰. In addition to P-values, comparisons were also scored by percentage identity. Percentage identity reflects the degree to which two segments of DNA or protein are identical over a particular length.

Example IX Trait summary for Transgenic Plants Overexpressing Sequences of the Invention

Appendix A, filed with priority U.S. patent application Ser. No. 09/713,994 on Nov. 16, 2000, provides traits observed when plants were modified to alter the expression of additional polynucleotide and polypeptide sequences.

The entire contents of Appendix A filed with priority U.S. patent application Ser. No. 09/713,994 are hereby incorporated by reference.

Table 5, below, provides a summary of the traits associated with prior filed Appendix A and the sequences of the invention. Each of the traits listed in Table 5 was observed to be modified in transgenic plants when the expression levels of each of these exemplary sequences were altered by overexpression of suppression. Table 5 lists the Gene IDentifier (GID) of each sequence, the SEQ ID NO: of the polynucleotide corresponding to the GID number, whether the sequence encoded by the respective GID was overexpressed or knocked out in plants, and the trait category and experimental observation made when the expression level of the respective GID was so altered.

TABLE 5 Sequences and traits observed when expression of the sequences was modified in plants DNA Overexpressor SEQ PRT (OE) or ID SEQ Knockout GID NO: ID NO: (KO) Trait Category Experimental Observation G4 111 112 OE Disease resistance Increased resistance to Botrytis G5 115 116 OE Plant size Small plant G6 119 120 G7 123 124 G9 48 128 OE Root morphology Increased root mass OE Salt tolerance Greater tolerance to 150 mM NaCl in a germination assay OE Sugar sensing/ More tolerant to glucose; greater sucrose tolerance germination and growth on 5% glucose medium OE Hormone Less sensitive to ABA; seedlings sensitivity were larger and greener in 0.5 μM ABA in a germination assay OE Cold tolerance More tolerant to cold; seedlings had less anthocyanin during growth in 8° C. G14 130 131 G19 3 135 OE Disease resistance Increased resistance to Erysiphe OE Hormon sensitivity Repressed by methyl jasmonate and induced by ACC G20 138 139 OE Seed sterols Increase in campesterol G22 19 143 OE Salt tolerance Greater tolerance to 150 mM NaCl in a germination assay G23 146 147 G25 150 151 OE Trichome Fewer trichomes at seedling stage OE Fusarium Expression induced by Fusarium infection G26 60 155 OE Sugar sensing/ Decreased germination and growth sucrose tolerance on 5% glucose medium G27 105 159 OE Plant size Abnormal development, small OE Altered C/N Increased sensitivity to media with sensing low nitrogen or lacking nitrogen source G28 5 163 OE Disease resistance Increased resistance to Botrytis OE Disease resistance Increased resistance to Erysiphe OE Disease resistance Increased resistance to Sclerotinia G29 166 167 G30 170 171 OE Leaf morphology Glossy green leaves OE Light response Increased shade tolerance; lack of shade avoidance phenotype G35 174 175 G36 178 179 G38 61 183 OE Sugar sensing/ Reduced germination on 5% sucrose tolerance glucose medium G39 186 187 G43 62 191 OE Sugar sensing/ Decreased germination and growth sucrose tolerance on 5% glucose medium G44 194 195 G142 198 199 OE Flowering time Early flowering G148 202 203 G152 206 207 G157 210 211 OE Flowering time Modest overexpression triggers early flowering; greater overexpression delays flowering G161 214 215 OE Altered C/N Increased sensitivity to media with sensing low nitrogen or lacking nitrogen source G164 218 219 G177 222 223 G178 226 227 G180 100 231 OE Seed oil content Decreased seed oil OE Flowering time Early flowering G187 56 235 OE Morphology Long, thin cotyledons at seedling stage; several flower abnormalities including strap-like, sepaloid petals G188 1 239 KO Salt and More tolerant to salt and/or osmotic hyperosmotic stress stress: better germination in 150 mM NaCl, 300 mM mannitol, 9.4% sucrose or 5% glucose KO Disease resistance Increased susceptibility to Fusarium G190 242 243 G192 91 247 OE Seed oil content Decreased seed oil content OE Flowering time Late flowering G194 250 251 OE Plant size Small plant OE Water deprivation More tolerant to desiccation tolerance G197 254 255 OE Seed oil content Increased seed oil OE Seed protein Decreased seed protein content G198 258 259 OE Salt tolerance More tolerant to salt; seedlings were larger and greener in a germination assay on 150 mM NaCl G200 262 263 KO Altered C/N Increased sensitivity to media with sensing low nitrogen or lacking nitrogen source G201 266 267 OE Seed protein Increased seed protein content content OE Seed oil content Decreased seed oil content G202 270 271 OE Seed protein Decreased seed protein content content OE Seed oil content Increased seed oil content G203 274 275 G204 279 G206 282 280 OE Seed size Large seeds G207 63 284 OE Sugar sensing/ Decreased germination on 5% glucose tolerance glucose medium KO Disease resistance Increased susceptibility to Botrytis OE Disease resistance Increased resistance to Erysiphe G208 102 288 OE Flowering time Early flowering G209 291 292 G210 295 296 G212 301 299 OE Altered trichome Partially to fully glabrous on initiation and adaxial surface of leaves number G214 35 303 OE Leaf fatty acids Increased leaf fatty acids OE Leaf prenyl lipids Increased leaf chlorophyll and carotenoids OE Flowering time Late flowering OE Seed prenyl lipids Increased seed lutein G215 308 306 G216 311 309 G217 312 313 OE Seed fatty acids Increase in 20:2 fatty acid in seeds G219 316 317 G220 320 321 G222 324 325 OE Seed oil content Decreased seed oil content OE Seed protein Increased seed protein content content G225 20 329 OE Root Increased root hairs OE Trichome Glabrous, lack of trichomes OE C/N sensing Greater growth and/or vigor on media with low nitrogen or lacking nitrogen source OE Nutrient uptake Increased tolerance to nitrogen- limited medium G226 21 333 OE Nutrient uptake Increased tolerance to nitrogen- limited medium OE Seed protein Increased seed protein content OE Root Increased root hairs OE Trichome Glabrous, lack of trichomes OE Sodium chloride More tolerant to salt; seedlings were larger and greener in a germination assay on 150 mM NaCl OE Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source G228 336 337 G229 36 341 OE Seed protein Decreased seed protein content OE Seed oil content Increased seed oil OE Other biochemistry Up-regulation of genes involved in secondary metabolism G231 74 345 OE Leaf fatty acids Increased leaf unsaturated fatty acids OE Seed protein Decreased seed protein content content OE Seed oil content Increased seed oil content G232 348 349 G233 37 353 OE Disease resistance Increased resistance to Botrytis OE Disease resistance Increased resistance to Erysiphe G234 92 357 OE Flowering time Late flowering OE Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source G237 7 361 OE Leaf biochemistry Increased leaf insoluble sugars OE Disease resistance Increased resistance to Erysiphe OE Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source G239 364 365 OE Expression/ABA Expression induced by 0.5 μM treatment ABA OE Expression from Expression induced by drought drought OE Expression/heat Expression induced by 32° C. treatment OE Expression/hyperosmotic Expression induced by stress hyperosmotic stress G241 368 369 OE Seed oil content Decreased seed oil KO Seed protein Altered seed protein content content OE Sugar sensing/ Decreased germination and growth glucose tolerance on 5% glucose medium G242 372 373 OE Leaf insoluble Increased arabinose sugars G245 376 377 G249 380 381 OE Flowering time Late flowering OE Time to senescence Delayed senescence G251 384 385 G252 388 389 G254 64 393 OE Sugar sensing/ Decreased germination and growth glucose tolerance on 5% glucose medium G256 22 397 OE Cold tolerance More tolerant to cold; increased seedling vigor and root growth at 8° C. in germination and growth assays G260 400 401 G261 4 404 OE Disease resistance Increased susceptibility to Botrytis G262 407 406 G263 65 409 OE Sugar sensing/ Decreased root growth on 9.4% glucose tolerance sucrose medium OE Tissue-specific Root specific expression expression G271 412 413 G273 415 416 G274 75 419 OE Leaf insoluble Increased leaf arabinose sugars G279 421 422 G285 424 425 G291 427 428 OE Seed oil content Increased seed oil content G306 430 431 OE Leaf insoluble Altered leaf insoluble sugars: sugars increased galactose, decreased arabinose, mannose, rhamnose and xylose G307 76 435 OE Sugar sensing/ No germination on 5% glucose glucose tolerance medium G308 66 439 G313 442 443 G315 446 447 G321 450 451 G322 453 454 G326 457 458 OE Altered C/N Increased sensitivity to media with sensing low nitrogen or lacking nitrogen source G328 461 462 G329 464 465 G330 467 468 OE Cell wall Xylose and rhamnose levels were composition elevated G335 473 471 G343 474 475 OE Glyphosate Increased glyphosate resistance resistance G345 477 478 G346 77 482 OE Leaf fatty acids Significant increase in 18:2 leaf fatty acid level OE Seed oil content Decreased seed oil G355 484 485 G357 488 489 OE Morphology and Most transformants died by the development flowering stage; potential herbicide target G361 93 493 OE Flowering time Late flowering G363 496 497 G364 500 501 OE Morphology and Most transformants died by the development flowering stage; potential herbicide target G368 504 505 G371 508 509 OE Disease resistance Increased susceptibility to Botrytis G376 511 512 G378 16 515 OE Disease resistance Increased resistance to Erysiphe G384 518 519 OE Altered C/N Increased sensitivity to media with sensing low nitrogen or lacking nitrogen source G385 521 522 OE Plant size Small plant OE Inflorescence Short inflorescence stems OE Leaf morphology Dark green plant G388 525 526 G389 529 530 G390 533 534 OE Flowering time Early flowering OE Plant morphology Abnormal, disorganized phyllotaxy; exhibited stem bifurcations in which shoot meristems split to form two or three separate shoots G391 537 538 OE Altered architecture Altered shoot development; T1 plants were dark green with short bolts, small leaves and short siliques G393 541 542 G394 33 546 OE Cold tolerance More sensitive to 8° C.; plants became chlorotic and leaves senesced prematurely G395 549 550 G396 552 553 G397 556 557 G398 560 561 G399 564 565 G400 567 568 G404 570 571 G409 8 574 OE Disease resistance Increased resistance to Erysiphe G411 576 577 G412 580 581 G414 584 585 G418 9 588 OE Disease resistance Increased resistance to Pseudomonas OE Seed protein Decreased seed protein content content G419 23 591 OE Low nutrient Increased tolerance to potassium- tolerance free medium G425 593 594 OE Disease resistance Increased resistance to Pseudomonas G426 596 597 G428 49 601 OE Leaf insoluble Increased leaf insoluble sugars sugars OE Leaf morphology Severe lobing of leaves conferring a parsley-like shape G431 55 605 OE Developmental Extremely deleterious or lethal defects G432 608 609 G435 612 613 OE Leaf insoluble Increased leaf insoluble sugars sugars G438 52 616 KO Altered architecture Reduced branching KO Stem lignification Reduced lignin OE Leaf morphology Increased leaf size; larger, flatter leaves OE Leaf morphology Altered leaf shape; broad flat leaves G439 619 620 G440 623 624 OE Disease resistance Increased resistance to Erysiphe G441 627 628 G442 631 632 G443 635 636 G444 639 640 G448 643 644 G449 647 648 G451 651 652 G452 655 656 G455 659 660 G456 663 664 OE Seed protein Decreased seed protein content OE Seed oil content Increased seed oil G459 666 667 G461 670 671 G462 673 674 G463 677 678 G464 24 682 OE Seed oil content Increased seed oil OE Heat tolerance More tolerance to heat; seedlings were larger and greener in germination and growth assays at 32° C. OE Leaf morphology Altered leaf shape OE Seed protein Decreased seed protein content content G466 685 686 G467 688 689 G470 57 692 OE Fertility Short stamen filaments; pollen produced, but not deposited on the stigma G474 695 696 G475 699 700 OE Flowering time Early flowering G481 703 704 OE Hyperosmotic More tolerant to mannitol; greater stress germination and growth on 300 mM mannitol medium OE Hormone Less sensitive to ABA; seedlings sensitivity were larger and greener in 0.5 μM ABA in a germination assay OE Sugar More tolerant to sucrose; better sensing/sucrose germination on 9.4% sucrose tolerance medium OE Heat tolerance More tolerance to heat; seedlings were larger and greener in germination and growth assays at 32° C. OE Water deprivation More tolerant to desiccation and tolerance drought G482 25 708 OE Salt tolerance More tolerant to salt; seedlings were larger and greener in a germination assay on 150 mM NaCl OE Hyperosmotic More tolerant to mannitol; greater stress germination and growth on 300 mM mannitol medium OE Heat tolerance More tolerance to heat; seedlings were larger and greener in germination and growth assays at 32° C. OE Water deprivation Increased survival and recovery tolerance from drought G483 711 712 OE Water deprivation Better recovery from drought tolerance G484 714 715 KO Altered seed Altered glucosinolate profile glucosinolates OE Water deprivation More tolerant to desiccation tolerance G485 718 719 OE Flowering time Early flowering KO Flowering time Late flowering OE Water deprivation Increased survival and recovery tolerance from drought OE Salt tolerance More tolerant to salt; seedlings were larger and greener in a germination assay on 150 mM NaCl OE Cold tolerance Greater tolerance to cold; in 8° C. in germination and growth assays; seedlings were larger and greener during germination and larger during growth OE Hormone Less sensitive to ABA; seedlings sensitivity were larger and greener in 0.5 μM ABA in a germination assay OE Sugar More tolerant to sucrose; better sensing/sucrose germination on 9.4% sucrose tolerance medium G486 94 723 OE Flowering time Late flowering OE Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source G489 34 727 OE Hyperosmotic More tolerant to mannitol; greater stress germination and growth on 300 mM mannitol medium OE Cold tolerance Greater tolerance to cold; in 8° C. in germination and growth assays; seedlings were larger, greened and had less anthocyanin during germination and growth OE Water deprivation More tolerant to desiccation and tolerance drought G501 730 731 G502 26 734 KO Hyperosmotic Increased sensitivity to 5% glucose stress or 150 mM NaCl G503 737 738 G505 743 741 OE Altered C/N Greater sensitivity to media with sensing low nitrogen or lacking nitrogen source G508 744 745 G509 748 749 KO Seed oil content Increased seed oil content KO Seed protein Decreased seed protein content content OE Seed glucosinolates Increased M39489 and M39497 G511 751 752 G513 755 756 G514 759 760 G516 764 762 OE Hyperosmotic More tolerant to mannitol; greater stress germination and growth on 300 mM mannitol medium OE Cold tolerance Increased tolerance to cold; in an 8° C. growth assay; seedlings had less anthocyanin OE Seed morphology Seeds of one line larger OE Plant size Seedlings of several lines larger G523 765 766 G524 769 770 G525 11 774 OE Disease resistance Increased tolerance to Pseudomonas OE Leaf insoluble Increased leaf insoluble sugars sugars G526 27 778 OE Tolerance to Increased sensitivity to 300 mM hyperosmotic stress mannitol or 10% polyethylene glycol G528 781 782 G529 785 786 G531 788 789 G532 792 793 G533 796 797 G535 800 801 G536 67 805 OE Sugar sensing/ Decreased germination and growth glucose tolerance on 5% glucose medium G560 838 839 G561 28 843 OE Seed oil content Increased seed oil content OE Nutrient uptake Increased tolerance to potassium- free medium G562 97 847 OE Flowering time Late flowering G563 850 851 G564 854 855 G566 858 859 G569 17 863 OE Defense gene Several genes repressed by G569 expression overexpression, including PR-1, MtN21 (upregulated by Erysiphe), PAR-1b (inducible by sucrose and salicylic acid), drought-induced protein Dr4, WAK 1 (upregulated by Erysiphe), antifungal protein, a glycine rich protein, polygalacturonase, putative β- expansin, flavonol synthase and pathogen-inducible protein CXc750. 12-oxo-phytodienoate 10,11-reductase (octadecanoid biosynthesis), lipoxygenase, GST and allene oxide synthase were consistently induced G570 866 867 G571 53 871 KO Time to senescence Delayed senescence KO Flowering time Late flowering G572 873 874 OE Disease resistance Increased resistance to Erysiphe G573 877 878 G575 881 882 G579 885 886 G582 889 890 G584 44 894 OE Seed morphology Large seeds G586 897 898 G589 900 901 G590 904 905 KO Seed oil content Increased seed oil content OE Flowering time Early flowering OE Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source G591 10 909 OE Disease resistance Increased resistance to Erysiphe OE Disease resistance Increased resistance to Botrytis OE Disease resistance Increased resistance to Sclerotinia OE Flowering time Late flowering G592 101 913 OE Flowering time Early flowering G593 916 917 G595 920 921 G598 78 925 OE Seed oil content Increased seed oil OE Leaf insoluble Altered insoluble sugars; increased sugars galactose levels G599 928 929 OE Leaf morphology Extreme rolling and curling of rosette leaves, giving the rosettes a pinwheel-like appearance G603 932 933 G605 79 937 OE Leaf fatty acids Altered leaf fatty acid composition; decreased 18:3 and increased 16:0 G607 940 941 G610 944 945 G615 58 949 OE Altered architecture Some plants were bushy and/or had fused cotyledons OE Fertility Little or no pollen production, poor filament elongation G616 2 953 OE Disease resistance Increased resistance to Erysiphe G629 956 957 OE Leaf morphology Altered leaf morphology OE Seed oil content Decreased seed oil content OE Seed protein Increased seed protein content content G630 959 960 OE Seed protein Increased seed protein content content OE Tissue-specific Embryo specific expression expression G632 962 963 G633 965 966 G634 969 970 OE Trichome Increased trichome density and size morphology and number OE Altered light Increased shade tolerance; lack of response and/or shade avoidance phenotype shade tolerance OE Water deprivation More tolerant to desiccation and tolerance drought G640 972 973 G641 975 976 G642 979 980 G649 983 984 G653 987 988 OE Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source G654 991 992 G656 994 995 G658 998 999 G659 1002 1003 G660 1006 1007 G661 1010 1011 G663 38 1015 OE Seed oil content Decreased seed oil OE Seed protein Increased seed protein content OE Anthocyanins Increased anthocyanins in leaf, root, seed G664 29 1019 OE Cold tolerance Increased tolerance to cold; better germination at 8° C. G665 1022 1023 G666 1026 1027 G668 45 1031 OE Seed protein Increased seed protein content content OE Seed oil content Decreased seed oil content OE Seed morphology Reduced seed color G669 1035 OE Morphology Small, rounded leaf morphology and spindly bolts with low fertility G670 1036 1037 OE Plant size Small plant G671 1040 1041 OE Stem Altered inflorescence stem structure OE Flower Reduced petal abscission OE Leaf Altered leaf shape; true leaves curl down, secondary bolts replaced by odd leaf-like structures OE Size Small plant OE Fertility Reduced fertility/underdevelopment of flowers G672 1044 1045 G673 1048 1049 G675 1051 1052 G676 1055 1056 OE Trichome Reduced trichome number, ectopic trichome formation G677 1059 1060 G679 1063 1064 G680 68 1067 OE Flowering time Late flowering OE Sugar sensing/ Reduced germination on 5% glucose tolerance glucose medium G682 30 1071 OE Heat tolerance More tolerance to heat; seedlings were larger and greener in germination and growth assays at 32° C. OE Salt tolerance More tolerant to salt; seedlings were larger and greener in a germination assay on 150 mM NaCl OE Hyperosmotic More tolerant to mannose; more stress tolerant to sucrose; better germination on a 9.4% sucrose medium OE Hormone Less sensitive to ABA; seedlings sensitivity were larger and greener in 0.5 μM ABA in a germination assay OE Low nutrient More tolerant to nitrogen-limiting tolerance conditions OE Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source OE Water deprivation More tolerant to desiccation and tolerance drought OE Trichome number Glabrous, lack of trichomes OE Root morphology Increased root hair number G699 1074 1075 G713 1077 1078 G714 1080 1081 OE Leaf morphology Some lines had long, narrow, curled leaves G718 1084 1085 OE Seed protein Increased seed protein content OE Leaf fatty acids Altered leaf fatty acid composition; decreased 16:0 and 16:3, increased 16:1 and 18:3 OE Seed prenyl lipids Increased seed lutein OE Seed oil content Decreased seed oil OE Seed fatty acids Seed fatty acids; decrease in 18:1 fatty acids in seeds G721 1088 1089 G723 1092 1093 G725 1096 1097 G726 1100 1101 G727 1103 1104 G729 1107 1108 G731 1111 1112 G732 47 1116 OE Seed protein One OE line had increased, another content decreased seed protein content OE Seed oil content One OE line had increased, another decreased seed oil content OE Altered architecture Reduced apical dominance OE Flower morphology Abnormal flowers G735 1119 1120 G736 98 1123 OE Flowering time Late flowering OE Leaf morphology Small, dark green rounded leaves with long petioles G740 106 1126 OE Altered growth rate Slow growth G743 1129 1130 G748 54 1134 OE Stem More vascular bundles in stem OE Flowering time Late flowering OE Seed prenyl lipids Increased lutein content G749 1137 1138 G751 1141 1142 G752 1144 1145 OE Flowering time Late flowering G759 1147 1148 G763 1150 1151 G764 1153 1154 G773 1156 1157 OE Altered C/N Increased sensitivity media with sensing low nitrogen or lacking nitrogen source G776 39 1161 OE Seed oil Altered seed fatty acid composition; composition decreased 20:1 and 22:1 fatty acids G777 80 1165 OE Seed oil content Decreased seed oil OE Leaf insoluble Increased leaf rhamnose sugars G778 40 1169 OE Seed oil Increased seed 18:1 fatty acid composition G779 1172 1173 OE Fertility Reduced fertility OE Flower Homeotic transformations; conversion of sepals to carpels, most severely affected showed full conversion of sepals to carpels with ovules, stigmatic tissue on Petals and stamens, and in some cases showed organ fusions G780 1176 1177 KO Seed fatty acids Significant increases in 16:0, 18:0, and 20:0 and decreases in 18:2, 20:1, and 20:2 OE Seed fatty acids Significant increase in 18:2 and a significant decrease in 18:3 G782 1180 1181 OE Sugar sensing/ More tolerant to sucrose; better sucrose tolerance germination on 9.4% sucrose medium G783 1184 1185 OE Sugar sensing/ More tolerant to sucrose; better sucrose tolerance germination on 9.4% sucrose medium G784 1187 1188 G786 1191 1192 G787 1194 1195 G788 1198 1199 G791 1202 1203 OE Seed oil Decreased decrease in 18:1 seed composition fatty acid OE Leaf insoluble Altered leaf cell wall sugars polysaccharide composition OE Leaf fatty acid Decreased 18:2 leaf fatty acid composition G792 1206 1207 G793 1210 1211 OE Disease resistance Increased resistance to Sclerotinia G795 1214 1215 G798 1218 1219 G801 1221 1222 OE Salt tolerance More tolerant to salt; seedlings were larger and greener in a germination assay on 150 mM NaCl G802 1225 1226 G804 1228 1229 G811 1232 1233 G830 1235 1236 G832 1238 G849 1239 1240 KO Seed protein Altered seed protein content content KO Seed oil content Increased seed oil content KO Seed sterols Decease in β-sitosterol G860 1242 1243 G864 1246 1247 OE Plant size Small plant OE Cold tolerance Increased adult stage sensitivity to 8° C. OE Heat tolerance More tolerance to heat; seedlings were larger and greener in a germination assay at 32° C. G865 13 1251 OE Disease resistance Increased resistance to Erysiphe OE Disease resistance Increased susceptibility to Botrytis OE Flowering time Early flowering OE Seed protein Increased seed protein content OE Altered Numerous secondary inflorescence morphology meristems-bushy appearance G866 1253 1254 OE Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source G867 69 1258 OE Sugar sensing/ More tolerant to sucrose; better sucrose tolerance seedling vigor on 9.4% sucrose medium OE Salt tolerance More tolerant to salt; better seedling vigor in a germination assay on 150 mM NaCl OE Water deprivation Increased survival and recovery tolerance from drought OE Hormone Less sensitive to ABA; seedlings sensitivity were larger and greener in 0.5 μM ABA in a germination assay OE Cold tolerance Increased tolerance to cold; at 8° C. in germination and growth assays, some seedlings were larger and during germination and growth G869 6 1262 OE Leaf insoluble Increase in fucose sugars OE Seed oil Increased 18:1 seed fatty acids composition OE Leaf fatty acids 16:0 levels decreased and 16:3 levels increased OE Disease resistance Increased resistance to Erysiphe OE Morphology: other Small and spindly plant OE Flower morphology Abnormal anther development G872 1265 1266 KO Developmental Embryo lethal defects OE Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source G877 1268 1269 KO Embryo lethal Embryo lethal phenotype: potential herbicide target G881 14 1273 OE Disease resistance Increased susceptibility to Botrytis OE Disease resistance Increased susceptibility to Erysiphe G883 41 1277 OE Seed prenyl lipids Decreased seed lutein G886 1280 1281 G891 1284 1285 G896 15 110 KO Disease resistance Increased susceptibility to Fusarium OE Disease resistance Increased resistance to Botrytis G897 1292 1293 G899 1296 1297 G902 1299 1300 G908 1303 1304 G909 1307 1308 G911 31 1311 OE Low nutrient Increased growth on potassium-free tolerance medium OE Seed protein Increased seed protein content content OE Seed oil content Decreased seed oil content G912 70 1314 OE Freezing tolerance Increased freezing tolerance OE Altered Dark green color morphology OE Water deprivation Increased survival and recovery tolerance from drought OE Sugar sensing/ Reduced cotyledon expansion in glucose tolerance 5% glucose OE Plant size Small plant OE Flowering time Late flowering OE Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source G913 1317 1318 OE Flowering time Late flowering OE Freezing tolerance Increased freezing tolerance OE Water deprivation Increased survival and recovery tolerance from drought OE Cold tolerance Increased tolerance to cold; more tolerant to 8° C. in a growth assay; some seedlings had less anthocyanin G914 1321 1322 G915 1325 1326 G921 1329 1330 OE Hyperosmotic Increased sensitivity to 10% stress tolerance polyethylene glycol or 150 mM salt OE Leaf Serrated leaves G927 1333 1334 G928 1337 1338 OE Sugar More tolerant to sucrose; better sensing/sucrose germination on 9.4% sucrose tolerance medium OE Water deprivation More tolerant to desiccation tolerance OE Cold tolerance More tolerant to cold; in an 8° C. germination assay, seedlings were larger and had less anthocyanin G929 1341 1342 G932 1345 1346 OE Leaf morphology Altered development, dark green color OE Plant size Reduced size OE Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source G938 42 1350 OE Seed oil Overexpressors had increased 16:0, composition 18:0, 20:0, and 18:3 fatty acids, decreased 18:2, 20:1, 22:1 fatty acids G939 1353 1354 G941 1357 1358 G942 1363 1361 G960 1364 1365 G964 32 1369 OE Heat tolerance More tolerance to heat; seedlings were larger and greener in a germination assay at 32° C. KO Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source G965 1371 1372 OE Seed oil Increase in 18:1 fatty acid composition G975 84 1376 OE Leaf fatty acids Increased wax in leaves OE Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source OE Water deprivation Increased survival and recovery tolerance from drought G976 1379 1380 G977 1383 1384 OE Plant size Small plant OE Morphology: color Dark green OE Leaf morphology Altered leaf shape; generally wrinkled or curled OE Fertility Reduced fertility; underdeveloped flowers, abnormal inflorescences G986 1387 1388 G987 1391 1392 KO Leaf fatty acids Reduction in 16:3 fatty acid Presence of two xanthophylls, KO Leaf prenyl lipids tocopherol not normally found in leaves; reduced chlorophyll a and b G994 95 1396 OE Flowering time Late flowering OE Plant size Small plants G996 71 1400 OE Sugar sensing/ Reduced germination on 5% glucose tolerance glucose medium G997 1403 1404 G998 1407 1408 G1000 1413 1411 G1004 1414 1415 G1005 1418 1419 G1006 1422 1423 OE Disease resistance Increased resistance to Erysiphe OE Disease resistance Increased resistance to Sclerotinia G1008 1426 1427 OE Plant morphology Overexpressors were small and bushy G1017 1430 1431 G1020 1434 1435 OE Plant size Very small T1 plants G1021 1438 1439 G1025 1442 1443 G1030 1445 1446 G1034 1448 1449 G1038 51 1452 OE Leaf morphology Rounded leaves OE Leaf insoluble Decreased insoluble sugars sugars G1039 1454 1455 G1040 1458 1459 OE Seed morphology Smaller and more rounded seeds G1045 1462 1463 G1048 1466 1467 OE Erysiphe Increased tolerance to Erysiphe orontii OE Seed protein Increased seed protein content content OE Altered light Increased shade tolerance; lack of response and/or shade avoidance phenotype shade tolerance G1052 1469 1470 OE Flowering time Late flowering OE Seed prenyl lipids Decrease in lutein and increase in xanthophyll 1 G1055 1473 1474 G1057 1476 1477 G1058 1480 1481 G1060 1484 G1061 1487 1488 G1065 1491 1492 G1067 1494 1495 OE Leaf morphology Upcurled rosette leaves OE Plant size Small plant OE Fertility Reduced fertility OE Water deprivation Increased survival and recovery tolerance from drought OE Hormone Less sensitive to ABA; seedlings sensitivity were larger and greener in 0.5 μM ABA in a germination assay G1068 72 1499 OE Sugar Reduced cotyledon expansion in sensing/glucose 5% glucose tolerance G1071 1502 1503 G1072 1506 1507 G1073 59 1511 OE Plant size Increased plant size OE Seed morphology Larger seeds; increased seed yield OE Leaf Serrated leaves OE Flowering time Flowering slightly delayed OE Salt tolerance More tolerant to salt; seedlings were larger and greener in a germination assay on 150 mM NaCl OE Sugar More tolerant to sucrose; better sensing/sucrose germination on 9.4% sucrose tolerance medium OE Hyperosmotic More tolerant to mannitol; greater stress germination and growth on 300 mM mannitol medium OE Water deprivation More tolerant to desiccation and tolerance drought G1075 1514 1515 OE Plant size Small plant OE Flower morphology Reduced or absent petals, sepals and stamens OE Fertility Reduced fertility OE Leaf morphology Pointed leaves in some seedlings; twisted or curled leaves and serrations in rosette stage G1078 1518 1519 G1082 1521 1522 G1083 1525 1526 G1090 1529 1530 OE Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source G1093 1532 1533 G1095 1536 1537 G1099 1540 1541 G1100 1544 1545 OE Altered light Increased shade tolerance; lack of response and/or shade avoidance phenotype shade tolerance G1107 1548 1549 G1109 1552 1553 G1130 1556 1557 G1131 1560 1561 G1133 81 1565 OE Leaf prenyl lipids Decreased leaf lutein G1134 1567 1568 OE Silique morphology Siliques with altered shape OE Hormone Altered response to ethylene: longer sensitivity hypocotyls and lack of apical hook OE Hormone Less sensitive to ABA; seedlings sensitivity were larger and greener in 0.5 μM ABA in a germination assay OE Root morphology Several seedlings had more root growth G1137 1571 1572 G1141 1575 1576 G1149 1578 1579 G1181 1582 1583 OE Plant size Small T1 plants G1196 1585 1586 G1197 1588 1589 G1202 1592 1593 OE Leaf fatty acids Significant increase (>2 standard deviation) in 18:0 and 18:1 fatty acids; decrease in 18:3 saturated fatty acids in leaves G1207 1595 1596 G1208 1600 1598 G1218 1601 1602 G1228 1606 1604 OE Plant size Reduced size G1232 1607 1608 G1233 1610 1611 G1240 1613 1614 G1241 1616 1617 G1249 1619 1620 G1258 1623 1624 G1261 1627 1628 G1266 82 1632 OE Leaf fatty acids Decreased 16:0, 18:2, increased 18:3 OE Disease resistance Increased resistance to Erysiphe OE Disease resistance Increased resistance to Botrytis OE Disease resistance Increased resistance to Sclerotinia OE Plant size Small plant OE Fertility Reduced fertility OE Leaf insoluble Alterations in xylose, and mannose, sugars and galactose concentrations; decreased rhamnose, some lines had more arabinose OE Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source OE Salt tolerance More tolerant to salt; seedlings were larger and greener in a germination assay on 150 mM NaCl OE Hyperosmotic More tolerant to mannitol; greater stress germination and growth on 300 mM mannitol medium OE Hormone Less sensitive to ABA; seedlings sensitivity were larger and greener in 0.5 μM ABA in a germination assay OE Cold tolerance Increased tolerance to cold; in an 8° C. germination assay, seedlings were larger, greener and had less anthocyanin G1269 50 1636 OE Leaf morphology Long petioles, upturned leaves G1275 1639 1640 OE Plant size Small plant OE Altered architecture Reduced apical dominance OE Heat tolerance More tolerance to heat; seedlings were larger and greener in a germination assay at 32° C. OE Cold tolerance More tolerant to cold; in 8° C. germination and growth assays; some seedlings were larger and had less anthocyanin OE Hormone Less sensitive to ABA; seedlings sensitivity were larger and greener in 0.5 μM ABA in a germination assay G1293 1643 G1300 1644 1645 OE Seed fatty acids One line had a reduction in 16:0, 18:0 and 20:0 seed fatty acids and an increase in the unsaturated 18:1 and 18:2 fatty acids; another line had significant increases in 16:0, 18:0 and 20:0 fatty acids and a reduction in 20:1 G1309 1647 1648 OE Plant size Small plant OE Leaf insoluble Increased mannose sugars G1311 1651 1652 OE Fertility Reduced fertility OE Plant size Small plant G1315 1655 1656 OE Leaf chemistry Increased leaf β-carotene G1319 1659 1660 G1321 1663 1664 G1323 1667 1668 OE Seed oil content Decreased seed oil OE Seed protein Increased seed protein content OE Plant size Small T1 plants OE Morphology: color Dark green G1324 83 1672 OE Leaf prenyl lipids Decreased leaf lutein, increased leaf xanthophyll G1326 1675 1676 OE Flower morphology Petals and sepals were smaller OE Plant size Small plant OE Fertility Reduced fertility G1327 1679 1680 OE Leaf morphology Dark green leaves G1328 43 1684 OE Seed prenyl lipids Decreased seed lutein G1329 1687 1688 G1333 1691 1692 G1334 104 1696 OE Plant size Some lines were small OE Plant size Larger seedlings OE Leaf morphology Dark green leaves G1335 96 1700 OE Flowering time Late flowering OE Dev and morph Slow growth G1337 73 1704 OE Sugar sensing Decreased germination on 9.4% sucrose medium OE Leaf fatty acids Altered leaf fatty acid composition G1338 1706 1707 G1340 1710 1711 G1349 1714 1715 G1350 1718 1719 G1351 1721 1722 G1352 1725 1726 G1355 1728 1729 OE Seed oil content Reduced seed oil G1363 1732 1733 OE Disease resistance Increased resistance to Fusarium OE Water deprivation Increased tolerance to desiccation G1366 1736 1737 G1367 1740 1741 G1383 1744 1745 G1385 1748 1749 G1389 1752 1753 G1390 1756 1757 G1394 1760 1761 G1395 1764 1765 G1396 1768 1769 G1398 1772 1773 G1403 1776 1777 KO Seed fatty acids Increased 16:0 and 18:0 and decreased 20:2 seed fatty acids G1411 1780 1781 OE Altered architecture Loss of apical dominance G1416 1784 1785 G1419 1788 1789 OE Altered seed Increased seed protein protein G1427 1792 1793 G1435 99 1796 OE Flowering time Late flowering OE Plant size Increased plant size OE Leaf morphology Dark green leaves G1437 1799 1800 G1438 1802 1803 G1439 1805 1806 G1443 1808 1809 G1449 1811 1812 OE Seed protein Increased seed protein content content OE Flower morphology Larger flowers with more open petals; extra petals G1456 1815 1816 G1466 1819 1820 G1489 1823 1824 G1496 1826 1827 OE Altered seed oil Increased seed oil content G1499 1830 1831 OE Architecture Bolts terminating without an inflorescence; in some lines, flowers replaced with filamentous structures or carpelloid structures; less severely affected lines produced flowers where sepals were converted to carpelloid tissue OE Flower morphology Petals and stamens were absent or reduced in size and number OE Morphology: other Dark green leaves G1509 1834 1835 G1514 1837 1838 OE Disease resistance Increased susceptibility to Botrytis G1518 1840 1841 G1519 1843 1844 KO Embryo lethal Embryo lethal phenotype: potential herbicide target G1526 1848 1846 KO Altered seed oil Increased seed oil content G1528 1849 1850 G1537 1852 1853 G1538 1856 1857 G1540 1860 1861 OE Cell differentiation Reduced cell differentiation in meristem G1541 1864 1865 G1542 1868 1869 G1543 1872 1873 OE Altered architecture Compact plant OE Morphology: color Dark green color OE Seed oil content Decreased seed oil OE Altered leaf prenyl Increase in chlorophyll a and b lipids G1550 1876 1877 G1586 1880 1881 OE Leaf morphology Narrow leaves G1634 1884 1885 OE Seed protein Decreased seed protein content content OE Seed oil content Increased seed oil G1635 1888 1889 OE Morphology Primary transformant had reduced apical dominance, reduced bolt elongation, narrow rosette leaves, and poor fertility G1636 1892 1893 G1638 1896 1897 G1640 1900 1901 OE Seed oil content Increased seed oil G1643 1906 1904 G1646 1907 1908 OE Seed oil content Increased seed oil OE Cold tolerance More tolerant to cold; in an 8° C. germination assay, some seedlings were larger and had less anthocyanin OE Water deprivation More tolerant to desiccation G1650 1911 1912 G1653 1915 1916 G1655 1919 1920 G1664 1923 1924 G1667 1927 1928 OE Seed protein Increased seed protein content content OE Seed oil content Decreased seed oil OE Leaf prenyl lipids Increased β-carotene G1669 1931 1932 G1699 1934 1935 G1705 1938 1939 G1742 1942 1943 G1773 1947 KO Altered C/N Greater growth and/or vigor on sensing media with low nitrogen or lacking nitrogen source G1785 1948 1949 G1787 1953 G1807 1955 OE Oxidative stress More sensitive to acifluorfen G1836 1956 1957 OE Salt tolerance More tolerant to salt; seedlings were larger and greener in a germination assay on 150 mM NaCl OE Sugar More tolerant to sucrose; better sensing/sucrose germination on 9.4% sucrose tolerance medium OE Hormone Less sensitive to ABA; seedlings sensitivity were larger and greener in 0.5 μM ABA in a germination assay OE Cold tolerance More tolerant to cold; in an 8° C. germination assay; seedlings were larger, greener and had less anthocyanin OE Water deprivation Increased survival and recovery tolerance from drought OE Flowering time Some lines slightly early flowering G1894 1960 1961 G1900 1963 1964 OE Flowering time Late flowering G1901 1966 1967 G1903 1969 1970 OE Seed protein Decreased seed protein content content G1911 1972 1973 G1917 1976 1977 KO Seed glucosinolate Significant increase in peak M39489 G2019 1979 KO Leaf prenyl lipids Significant (>2 standard deviation) increase in the leaf prenyl lipids, xanthophylls G2484 1980 1981

Example X Plants Overexpressing G1435 (SEQ ID NOs: 99 and 1796; a GARP Family Transcription Factor)

G1435 (SEQ ID NO: 99 and polypeptide SEQ ID NO: 1796) was isolated as a cDNA clone. G1435 was later identified in the sequence of BAC F2015, GenBank accession number AB025604, released by the Arabidopsis Genome Initiative.

Experimental observations. The complete sequence of G1435 was determined. The function of this gene was analyzed using transgenic plants in which G1435 was expressed under the control of the 35S promoter. Plants overexpressing G1435 were larger than wild-type controls, and had dark green leaves. Primary transformants were late flowering. G1435 was expressed throughout the plant, though at lower levels in roots and germinating seeds. It is not significantly induced or repressed by any condition tested.

A second experiment in which 35S::G1435 plants were grown confirmed the late flowering phenotype of three T2 lines. G1435 could thus be used to influence flowering time in crop plants. In species such as sugarbeet where the vegetative parts of the plants constitute the crop and the reproductive tissues are discarded, it would be advantageous to delay or prevent flowering. Extending vegetative development could bring about large increases in yields.

Another potential indicator of increased yield conferred by G1435 overexpression was the increased plant size and green color (suggesting increased photosynthetic capacity). It was thus expected that G1435 may be useful for increasing yield and/or crop quality in crops, including where the vegetative portion of the plant is harvested. A confirmation of an increase in yield in a commercially important plant species was provided when a field trial of corn plants overexpressing G1435 showed two lines with significantly increased broad acre yield relative to negative segregant controls (Table 6). These lines, 642 and 653, showed increased total kernel number and total kernel weight. Line 653 showed the higher percentage increase (a statistically significant increase) in photosynthesis compared to the negative segregant controls.

TABLE 6 Results in field trials comparing means of yield of transgenics to negative segregant controls Mean yield, Mean yield, Percent Line overexpressor control Difference difference p value 641 213.418 226.181 −12.763 −5.643 0.002 665 223.890 226.181 −2.291 −1.013 0.586 649 224.233 226.181 −1.948 −0.862 0.644 662 228.127 226.181 1.946 0.860 0.644 660 221.525 226.181 −4.656 −2.059 0.269 646 215.764 226.181 −10.417 −4.606 0.014 656 227.422 226.181 1.241 0.548 0.769 664 226.313 226.181 0.132 0.058 0.975  642* 232.418 226.181 6.237 2.757 0.138  653* 234.244 226.181 8.063 3.565 0.056 654 229.372 226.181 3.191 1.410 0.448 651 230.383 226.181 4.202 1.858 0.319 *Increased yield in these lines observed (p < 0.15)

Example XI Utilities of G1435 (SEQ ID NOs: 99 and 1796) and its Phylogenetically-Related Sequences

Based on the data obtained in the above-disclosed Example, the darker green color, increased photosynthesis, increased plant size and increased yield of G1435 overexpressors all indicate that G1435-related sequence overexpression can directly result in improved yield of crop plants, ornamental plants, and woody plants used in the food, ornamental, paper, pulp, lumber or other industries.

The invention thus includes G1435-overexpressing plants, and methods for producing G1435-overexpressing plants, or delaying flowering, increasing size, increasing photosynthesis, or increasing yield in a plant where the plant overexpresses G1435 or a phylogenetically and functionally-related sequence.

Example XII Transformation of Dicots to Produce Increased Photosynthesis, Yield or Stress Tolerance

Crop species that overexpress polypeptides of the invention may produce plants with increased photosynthetic capacity and/or yield, and/or increased tolerance to water deprivation, cold and/or nutrient tolerance in both stressed and non-stressed conditions. Thus, polynucleotide sequences listed in the Sequence Listing recombined into, for example, one of the expression vectors of the invention, or another suitable expression vector, may be transformed into a plant for the purpose of modifying plant traits for the purpose of improving yield and/or quality. The expression vector may contain a constitutive, tissue-specific or inducible promoter operably linked to the polynucleotide. The cloning vector may be introduced into a variety of plants by means well known in the art such as, for example, direct DNA transfer or Agrobacterium tumefaciens-mediated transformation. It is now routine to produce transgenic plants using most dicot plants (see Weissbach and Weissbach (1989) Methods for Plant Molecular Biology, Academic Press; Gelvin et al. (1990) Plant Molecular Biology Manual, Kluwer Academic Publishers; Herrera-Estrella et al. (1983) Nature 303: 209; Bevan (1984) Nucleic Acids Res. 12: 8711-8721; and Klee (1985) Bio/Technology 3: 637-642). Methods for analysis of traits are routine in the art and examples are disclosed above.

Numerous protocols for the transformation of tomato and soy plants have been previously described, and are well known in the art. Gruber et al. (1993), in Glick and Thompson (1993) Methods in Plant Molecular Biology and Biotechnology, eds., CRC Press, Inc., Boca Raton, describe several expression vectors and culture methods that may be used for cell or tissue transformation and subsequent regeneration. For soybean transformation, methods are described by Miki et al. (1993) in Methods in Plant Molecular Biology and Biotechnology, p. 67-88, Glick and Thompson, eds., CRC Press, Inc., Boca Raton; and U.S. Pat. No. 5,563,055, (Townsend and Thomas), issued Oct. 8, 1996.

There are a substantial number of alternatives to Agrobacterium-mediated transformation protocols, other methods for the purpose of transferring exogenous genes into soybeans or tomatoes. One such method is microprojectile-mediated transformation, in which DNA on the surface of microprojectile particles is driven into plant tissues with a biolistic device (see, for example, Sanford et al. (1987) Part. Sci. Technol. 5:27-37; Sanford (1993) Methods Enzymol. 217: 483-509; Christou et al. (1992) Plant. J. 2: 275-281; Klein et al. (1987) Nature 327: 70-73; U.S. Pat. No. 5,015,580 (Christou et al), issued May 14, 1991; and U.S. Pat. No. 5,322,783 (Tomes et al.), issued Jun. 21, 1994).

Alternatively, sonication methods (see, for example, Zhang et al. (1991) Bio/Technology 9: 996-997); direct uptake of DNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol or poly-L-ornithine (see, for example, Hain et al. (1985) Mol. Gen. Genet. 199: 161-168; Draper et al. (1982) Plant Cell Physiol. 23: 451-458); liposome or spheroplast fusion (see, for example, Deshayes et al. (1985) EMBO J., 4: 2731-2737; Christou et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3962-3966); and electroporation of protoplasts and whole cells and tissues (see, for example, Donn et al. (1990) in Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38: 53; D'Halluin et al. (1992) Plant Cell 4: 1495-1505; and Spencer et al. (1994) Plant Mol. Biol. 24: 51-61) have been used to introduce foreign DNA and expression vectors into plants.

After a plant or plant cell is transformed (and the latter regenerated into a plant), the transformed plant may be crossed with itself or a plant from the same line, a non-transformed or wild-type plant, or another transformed plant from a different transgenic line of plants. Crossing provides the advantages of producing new and often stable transgenic varieties. Genes and the traits they confer that have been introduced into a tomato or soybean line may be moved into distinct line of plants using traditional backcrossing techniques well known in the art. Transformation of tomato plants may be conducted using the protocols of Koornneef et al (1986) In Tomato Biotechnology: Alan R. Liss, Inc., 169-178, and in U.S. Pat. No. 6,613,962, the latter method described in brief here. Eight day old cotyledon explants are precultured for 24 hours in Petri dishes containing a feeder layer of Petunia hybrida suspension cells plated on MS medium with 2% (w/v) sucrose and 0.8% agar supplemented with 10 μM α-naphthalene acetic acid and 4.4 μM 6-benzylaminopurine. The explants are then infected with a diluted overnight culture of Agrobacterium tumefaciens containing an expression vector comprising a polynucleotide of the invention for 5-10 minutes, blotted dry on sterile filter paper and cocultured for 48 hours on the original feeder layer plates. Culture conditions are as described above. Overnight cultures of Agrobacterium tumefaciens are diluted in liquid MS medium with 2% (w/v/) sucrose, pH 5.7) to an OD₆₀₀ of 0.8.

Following cocultivation, the cotyledon explants are transferred to Petri dishes with selective medium comprising MS medium with 4.56 μM zeatin, 67.3 μM vancomycin, 418.9 μM cefotaxime and 171.6 μM kanamycin sulfate, and cultured under the culture conditions described above. The explants are subcultured every three weeks onto fresh medium. Emerging shoots are dissected from the underlying callus and transferred to glass jars with selective medium without zeatin to form roots. The formation of roots in a kanamycin sulfate-containing medium is a positive indication of a successful transformation.

Transformation of soybean plants may be conducted using the methods found in, for example, U.S. Pat. No. 5,563,055 (Townsend et al., issued Oct. 8, 1996), described in brief here. In this method soybean seed is surface sterilized by exposure to chlorine gas evolved in a glass bell jar. Seeds are germinated by plating on 1/10 strength agar solidified medium without plant growth regulators and culturing at 28° C. with a 16 hour day length. After three or four days, seed may be prepared for cocultivation. The seedcoat is removed and the elongating radicle removed 3-4 mm below the cotyledons.

Overnight cultures of Agrobacterium tumefaciens harboring the expression vector comprising a polynucleotide of the invention are grown to log phase, pooled, and concentrated by centrifugation. Inoculations are conducted in batches such that each plate of seed was treated with a newly resuspended pellet of Agrobacterium. The pellets are resuspended in 20 ml inoculation medium. The inoculum is poured into a Petri dish containing prepared seed and the cotyledonary nodes are macerated with a surgical blade. After 30 minutes the explants are transferred to plates of the same medium that has been solidified. Explants are embedded with the adaxial side up and level with the surface of the medium and cultured at 22° C. for three days under white fluorescent light. These plants may then be regenerated according to methods well established in the art, such as by moving the explants after three days to a liquid counter-selection medium (see U.S. Pat. No. 5,563,055).

The explants may then be picked, embedded and cultured in solidified selection medium. After one month on selective media transformed tissue becomes visible as green sectors of regenerating tissue against a background of bleached, less healthy tissue. Explants with green sectors are transferred to an elongation medium. Culture is continued on this medium with transfers to fresh plates every two weeks. When shoots are 0.5 cm in length they may be excised at the base and placed in a rooting medium.

Example XIII Transformation of Monocots to Produce Increased Photosynthesis Yield or Abiotic Stress Tolerance

Cereal plants such as, but not limited to, corn, wheat, rice, sorghum, or barley, may be transformed with the present polynucleotide sequences, including monocot or dicot-derived sequences such as those presented in the present Tables, cloned into a vector such as pGA643 and containing a kanamycin-resistance marker, and expressed constitutively under, for example, the CaMV 35S or COR15 promoters, or with tissue-specific or inducible promoters. The expression vectors may be one found in the Sequence Listing, or any other suitable expression vector may be similarly used. For example, pMEN020 may be modified to replace the NptII coding region with the BAR gene of Streptomyces hygroscopicus that confers resistance to phosphinothricin. The KpnI and BglII sites of the Bar gene are removed by site-directed mutagenesis with silent codon changes.

The cloning vector may be introduced into a variety of cereal plants by means well known in the art including direct DNA transfer or Agrobacterium tumefaciens-mediated transformation. The latter approach may be accomplished by a variety of means, including, for example, that of U.S. Pat. No. 5,591,616, in which monocotyledon callus is transformed by contacting dedifferentiating tissue with the Agrobacterium containing the cloning vector.

The sample tissues are immersed in a suspension of 3×10⁻⁹ cells of Agrobacterium containing the cloning vector for 3-10 minutes. The callus material is cultured on solid medium at 25° C. in the dark for several days. The calli grown on this medium are transferred to Regeneration medium. Transfers are continued every 2-3 weeks (2 or 3 times) until shoots develop. Shoots are then transferred to Shoot-Elongation medium every 2-3 weeks. Healthy looking shoots are transferred to rooting medium and after roots have developed, the plants are placed into moist potting soil.

The transformed plants are then analyzed for the presence of the NPTII gene/kanamycin resistance by ELISA, using the ELISA NPTII kit from 5Prime-3Prime Inc. (Boulder, Colo.).

It is also routine to use other methods to produce transgenic plants of most cereal crops (Vasil (1994) Plant Mol. Biol. 25: 925-937) such as corn, wheat, rice, sorghum (Cassas et al. (1993)), and barley (Wan and Lemeaux (1994)). DNA transfer methods such as the microprojectile method can be used for corn (Fromm et al. (1990); Gordon-Kamm et al. (1990); Ishida (1990)), wheat (Vasil et al. (1992) Bio/Technol. 10:667-674; Vasil et al. (1993) Bio/Technol. 11: 1553-1558; Weeks et al. (1993) Plant Physiol. 102: 1077-1084), and rice (Christou (1991) Bio/Technol. 9:957-962; Hiei et al. (1994) Plant J. 6:271-282; Aldemita and Hodges (1996) Planta 199: 612-617; and Hiei et al. (1997) Plant Mol. Biol. 35:205-218). For most cereal plants, embryogenic cells derived from immature scutellum tissues are the preferred cellular targets for transformation (Hiei et al. (1997) supra; Vasil (1994) supra). For transforming corn embryogenic cells derived from immature scutellar tissue using microprojectile bombardment, the A188XB73 genotype is the preferred genotype (Fromm et al. (1990) Bio/Technol. 8: 833-839; Gordon-Kamm et al. (1990) Plant Cell 2: 603-618). After microprojectile bombardment the tissues are selected on phosphinothricin to identify the transgenic embryogenic cells (Gordon-Kamm et al. (1990) supra). Transgenic plants are regenerated by standard corn regeneration techniques (Fromm et al. (1990) supra; Gordon-Kamm et al. (1990) supra).

Example XIV Expression and Analysis of Increased Photosynthesis Yield or Abiotic Stress Tolerance in Non-Arabidopsis Species

It is expected that structurally similar orthologs of the G1435 clade of polypeptide sequences, including those found in the Sequence Listing, can confer increased yield relative to control plants. As sequences of the invention have been shown to increase photosynthesis and/or yield in a variety of plant species, it is also expected that these sequences will increase yield of crop or other commercially important plant species.

Northern blot analysis, RT-PCR or microarray analysis of the regenerated, transformed plants may be used to show expression of a polypeptide or the invention and related genes that are capable of inducing abiotic stress tolerance, and/or larger size.

After a dicot plant, monocot plant or plant cell has been transformed (and the latter regenerated into a plant) and shown to have greater size, improved yield, or able to tolerate greater planting density with a coincident increase in yield, the transformed monocot plant may be crossed with itself or a plant from the same line, a non-transformed or wild-type monocot plant, or another transformed monocot plant from a different transgenic line of plants.

The function of specific polypeptides of the invention, including closely-related orthologs, have been analyzed and may be further characterized and incorporated into crop plants. The ectopic overexpression of these sequences may be regulated using constitutive, inducible, or tissue specific regulatory elements. Genes that have been examined and have been shown to modify plant traits (including increasing yield and/or abiotic stress tolerance) encode polypeptides found in the Sequence Listing. In addition to these sequences, it is expected that newly discovered polynucleotide and polypeptide sequences closely related to polynucleotide and polypeptide sequences found in the Sequence Listing can also confer alteration of traits in a similar manner to the sequences found in the Sequence Listing, when transformed into any of a considerable variety of plants of different species, and including dicots and monocots. The polynucleotide and polypeptide sequences derived from monocots (e.g., the rice sequences) may be used to transform both monocot and dicot plants, and those derived from dicots (e.g., the Arabidopsis and soy genes) may be used to transform either group, although it is expected that some of these sequences will function best if the gene is transformed into a plant from the same group as that from which the sequence is derived.

Sequences of the invention, that is, members of the G1435 clade, may also be used to generate transgenic plants that have increased photosynthetic capacity, produce larger plants and/or greater yield than control plants.

It is expected that the same methods may be applied to identify other useful and valuable sequences of the present polypeptide clades, and the sequences may be derived from a diverse range of species.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The present invention is not limited by the specific embodiments described herein. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. Modifications that become apparent from the foregoing description and accompanying figures fall within the scope of the claims. 

1. A transgenic plant transformed with an expression vector comprising a polynucleotide sequence encoding a polypeptide having a conserved domain that has at least 85% sequence identity to amino acids 145-213 of SEQ ID NO: 163, wherein a plant transformed with the expression vector exhibits increased resistance to a fungal pathogen when compared to a control plant.
 2. The transgenic plant of claim 1, wherein the conserved domain is at least 91% identical to the conserved domain of amino acids 145-213 of SEQ ID NO:
 163. 3. The transgenic plant of claim 1, wherein the conserved domain is at least 95% identical to amino acids 145-213 of SEQ ID NO:
 163. 4. The transgenic plant of claim 1, wherein the conserved domain is at least 97% identical to amino acids 145-213 of SEQ ID NO:
 163. 5. The transgenic plant of claim 1, wherein expression of the polypeptide is regulated by a constitutive, inducible, or tissue-specific promoter.
 6. The transgenic plant of claim 1, wherein the fungal pathogen is Botrytis.
 7. The transgenic plant of claim 1, wherein the fungal pathogen is Erysiphe.
 8. The transgenic plant of claim 1, wherein the fungal pathogen is Sclerotinia.
 9. The transgenic plant of claim 1, wherein the transgenic plant is a transgenic seed comprising the expression vector of claim
 1. 10. The transgenic plant of claim 1, wherein the transgenic plant produces a greater yield than the control plant.
 11. A method for producing a transgenic plant having greater resistance to a fungal pathogen than a control plant comprising transforming a target plant with a recombinant construct encoding a polypeptide having a conserved domain at least 85% identical to amino acids 145-213 of SEQ ID NO: 163, wherein a plant transformed with the recombinant construct exhibits increased resistance to a fungal pathogen when compared to the control plant.
 12. The method of claim 11, wherein the conserved domain is at least 91% identical to amino acids 145-213 of SEQ ID NO:
 163. 13. The method of claim 11, wherein the conserved domain is at least 95% identical to amino acids 145-213 of SEQ ID NO:
 163. 14. The method of claim 11, wherein the conserved domain is at least 97% identical to amino acids 145-213 of SEQ ID NO:
 163. 15. The method of claim 11, wherein expression of the polypeptide is regulated by a constitutive, inducible, or tissue-specific promoter.
 16. The method of claim 11, wherein the fungal pathogen is Botrytis.
 17. The method of claim 11, wherein the fungal pathogen is Erysiphe.
 18. The method of claim 11, wherein the fungal pathogen is Sclerotinia.
 19. The method of claim 11, wherein the transgenic plant produces a greater yield than the control plant.
 20. A transgenic plant comprising a recombinant polynucleotide encoding a polypeptide with at least 85% amino acid sequence identity to SEQ ID NO:163, and wherein a plant transformed with the recombinant polynucleotide exhibits increased resistance to a fungal pathogen when compared to a control plant.
 21. The transgenic plant of claim 20, wherein the polypeptide is at least 90% identical to SEQ ID NO:
 163. 22. The transgenic plant of claim 20, wherein the polypeptide is at least 95% identical to SEQ ID NO:
 163. 23. A method for producing a transgenic plant having greater resistance to a disease than a control plant, the method comprising the step of transforming a target plant with a recombinant construct encoding a polypeptide that is at least 85% identical to SEQ ID NO: 163, wherein a plant transformed with the recombinant construct exhibits increased resistance to a fungal pathogen when compared to the control plant.
 24. The method of claim 23, wherein the polypeptide is at least 90% identical to SEQ ID NO:
 163. 25. The method of claim 23, wherein the polypeptide is at least 95% identical to SEQ ID NO:
 163. 